Anti human Notch4 antibody

ABSTRACT

The present invention provides an anti-human Notch4 antibody or a Notch4 binding fragment thereof that may have neutralizing activity against human Notch4, as well as a pharmaceutical composition comprising the same as the active ingredient. The present inventors obtained a mouse anti-human Notch4 antibody that has high neutralizing activity and binding affinity towards human Notch4 and determined the complementarity determining region (CDR) sequence of the mouse anti-human Notch4 antibody. This enabled the production of a humanized antibody comprising the variable region of heavy and light chains as well as the CDR sequence of the mouse anti-human Notch4 antibody.

TECHNICAL FIELD

The present invention relates to an antibody that binds to human Notch4.

BACKGROUND ART

Notch is a molecule that contributes to the determination of fate of cells of various tissues, and is indicated to be involved in e.g. differentiation, proliferation, and survival during each stages of early developmental stage, embryonic stage, and after birth. Four types of receptors including Notch1, Notch2, Notch3, and Notch4 as well as five types of ligands including Jagged1, Jagged2, DLL1, DLL3, and DLL4 are reported as the Notch family. When a Notch receptor expressed on an adjacent cell binds with a Notch ligand, the NRR domain present in the lower extracellular domain of the receptor is cleaved by TACE, and due to the structural change of the intracellular domain thus caused, the intracellular domain is cleaved by γ secretase. The Notch Intracellular (NIC) domain formed as a result migrates into the nucleus, forms a heterodimer with transcription factor CSL, and target molecules such as the aHes family or the Hey family are induced and expressed. These downstream molecules further induce and express various genes, and as a result, the Notch signal contributes to e.g. the maintenance of stem cells or progenitor cells, differentiation, cell cycle arrest, and cell fate determination (Non-Patent Literature 1).

Notch is also known to be involved in tumor formation. Notch1 mutation due to t(7;9) chromosomal translocation was first reported as being related to the onset of pre-T cell acute lymphoblastic leukemia (T-ALL). Moreover, the genome insertion site of Mouse Mammary Tumor Virus (MMTV) which is a spontaneous tumor onset model is reported to be Int3 (Notch4 intracellular domain), and it is reported that epithelial cell cancer such as breast cancer or salivary gland cancer are induced in a transgenic mouse where Int3 was force expressed (Non-Patent Literature 2). Notch4 is also reported to be related to the oncogenesis, progression, or metastasis of breast cancer (Non-Patent Literature 3), melanoma (Non-Patent Literature 4), stomach cancer (Non-Patent Literature 5), B-cell acute lymphocytic leukemia (B-ALL) (Non-Patent Literature 6), chronic lymphocytic leukemia (CLL) (Non-Patent Literature 7), glioma (Non-Patent Literature 8), hepatocellular carcinoma (Non-Patent Literature 9), lung cancer (Non-Patent Literature 10), renal cancer (Non-Patent Literature 11), Kaposi's sarcoma (Non-Patent Literature 12), and the like in humans.

The Notch signal also contributes intratumoral neovascularization. Notch1 and Notch4 are expressed as Notch receptors in vascular endothelial cells, and the expression of DLL4 and Jagged1 are confirmed as ligands. Tip cells present at the tip of new blood vessels highly express DDL4 with VEGF stimulation, and blood vessels are extended by sending a signal to the Notch receptor of the adjacent Stalk cell. On the other hand, Jagged1 competes with DLL4 for the Notch receptor and inhibits the binding of DLL4 with the Notch receptor. Since the signal from Jagged1 is weak compared to that from DLL4, the Notch signal is suppressed by binding with Jagged1. The intensity of the Notch signal is adjusted by the spatially differing expression patterns of these two ligands to control neovascularization (Non-Patent Literature 13).

Production of a DLL4 inhibitory antibody has been reported, in which when the signal from DLL4 is inhibited with a DLL4 inhibitory antibody, immature angiogenesis without bloodstream is enhanced inside a tumor and inhibition of tumor proliferation is induced. This is a completely different phenomenon from when a VEGF inhibitor inhibits the proliferation of vascular endothelial cells to suppress angiogenesis, and the Notch signal is gathering attention as a novel target for neovascularization inhibitors (Non-Patent Literature 14).

CITATION LIST

-   [Non-Patent Literature 1] Radtke et al. (2004), Nature Immunology     5,247-53. -   [Non-Patent Literature 2] Jhappan et al. (1992), Genes Dev. 6,345-55 -   [Non-Patent Literature 3] Nagamatsu et al. (2014), Anticancer Res.     34, 69-80 -   [Non-Patent Literature 4] Hardy et al. (2010), Cancer Res. 70,     10340-50 -   [Non-Patent Literature 5] Qian et al. (2015), Mol Cell Biochem. 401,     165-74 -   [Non-Patent Literature 6] Nwabo Kamdje et al. (2011), Blood 118,     380-9 -   [Non-Patent Literature 7] Nwabo Kamdje et al. (2012), Blood Cancer     Journal 2, e73 -   [Non-Patent Literature 8] Dell'Albani et al. (2012), Neuro-Oncology     16, 204-16 -   [Non-Patent Literature 9] Ahn et al. (2013), Hepatobiliary Pancreat     Dis Int. 12, 286-94 -   [Non-Patent Literature 10] Justilien et al. (2012), PLoS ONE 7,     e35040 -   [Non-Patent Literature 11] Boo et al. (2009), J Pediatr Surg. 44,     2031-6 -   [Non-Patent Literature 12] Curry et al. (2005), Oncogene 24, 6333-44 -   [Non-Patent Literature 13] Benedito et al. (2009), Cell 137, 1124-35 -   [Non-Patent Literature 14] Ridgway et al. (2006), Nature 444, 1083-7

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The object of the present invention is to provide an anti-human Notch4 antibody or a Notch4 binding fragment thereof that may have neutralizing activity against human Notch4, as well as a pharmaceutical composition comprising the same as the active ingredient.

Means for Solving the Problems

As a result of extensive investigation to solve the above problems, the present inventors succeeded in obtaining a mouse anti-human Notch4 antibody that has high neutralizing activity and binding affinity towards human Notch4. Moreover, by determining the complementarity determining region (CDR) sequence of said mouse anti-human Notch4 antibody, the present inventors enabled the production of a humanized antibody comprising the variable region of heavy and light chains as well as the CDR sequence of said mouse anti-human Notch4 antibody to complete the present invention.

In other words, in one embodiment, the present invention relates to the following inventions.

(1) An anti-Notch4 antibody or a Notch4 binding fragment thereof comprising:

(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or 16;

(b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 17 or 18;

(c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 19;

(d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 20;

(e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 21; and

(f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 22.

(2) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains and is selected from any of the following (i)-(vii):

(i) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45,

(ii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45,

(iii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47,

(iv) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 49,

(v) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 51,

(vi) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45, and

(vii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47.

(3) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (4) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (5) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (6) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 49. (7) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 51. (8) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (9) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (2), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (10) The antibody or a Notch4 binding fragment thereof according to any one of (1)-(9), wherein the constant region of said heavy chain and the constant region of said light chain comprise a human antibody-derived sequence. (11) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (10), wherein the constant region of the heavy chain comprises the constant region of human IgG. (12) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (11), wherein said constant region of human IgG is the constant region of human IgG2. (13) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (12), wherein said constant region of human IgG2 has a mutation V234A and/or G237A. (14) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (10), wherein the lysine residue at the carboxy terminal of the constant region of said heavy chain is artificially removed. (15) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (10), wherein the constant region of said light chain comprises the constant region of human Igκ. (16) A pharmaceutical composition comprising the anti-Notch4 antibody or a Notch4 binding fragment thereof according to any one of (1)-(15). (17) The pharmaceutical composition according to (16) which further comprises a pharmaceutically acceptable carrier. (18) The pharmaceutical composition according to (17) which is used for treatment of non-small cell lung cancer. (19) The pharmaceutical composition according to (17) which is used for treatment of thyroid cancer. (20) The pharmaceutical composition according to (17) which is used for treatment of prostate cancer. (21) The pharmaceutical composition according to (17) which is used for treatment of hepatocellular carcinoma.

In other embodiments, the present invention also relates to the following inventions.

(1′) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains and is selected from any of the following (i)-(vii):

(i) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45,

(ii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45,

(iii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47,

(iv) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 49,

(v) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 51,

(vi) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45, and

(vii) an antibody in which the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47.

(2′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (3′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (4′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (5′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 49. (6′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 51. (7′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (8′) The anti-Notch4 antibody or a Notch4 binding fragment thereof according to (1′), wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (9′) The antibody or a Notch4 binding fragment thereof according to any one of (1′)-(8′), wherein the constant region of said heavy chain and the constant region of said light chain comprise a human antibody-derived sequence. (10′) The antibody or a Notch4 binding fragment thereof according to (9′), wherein the constant region of the heavy chain comprises the constant region of human IgG. (11′) The antibody or a Notch4 binding fragment thereof according to (10′), wherein said constant region of human IgG is the constant region of human IgG2. (12′) The antibody or a Notch4 binding fragment thereof according to (11′), wherein said constant region of human IgG2 has a mutation V234A and/or G237A. (13′) The antibody or a Notch4 binding fragment thereof according to (9′), wherein the lysine residue at the carboxy terminal of the constant region of said heavy chain is artificially removed. (14′) The antibody or a Notch4 binding fragment thereof according to (9′), wherein the constant region of said light chain comprises the constant region of human Igκ. (15′) A pharmaceutical composition comprising the anti-Notch4 antibody or a Notch4 binding fragment thereof according to any one of (1′)-(14′). (16′) The pharmaceutical composition according to (15′) which further comprises a pharmaceutically acceptable carrier. (17′) The pharmaceutical composition according to (16′) which is used for treatment of non-small cell lung cancer. (18′) The pharmaceutical composition according to (16′) which is used for treatment of thyroid cancer. (19′) The pharmaceutical composition according to (16′) which is used for treatment of prostate cancer. (20′) The pharmaceutical composition according to (16′) which is used for treatment of hepatocellular carcinoma.

In other embodiments, the present invention further relates to the following inventions.

(1″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (2″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (3″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (4″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 49. (5″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 33 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 51. (6″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 45. (7″) An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or a Notch4 binding fragment thereof comprises heavy and light chains, and the variable region of said heavy chain comprises the amino acid sequence of SEQ ID NO. 35 and the variable region of said light chain comprises the amino acid sequence of SEQ ID NO. 47. (8″) The antibody or a Notch4 binding fragment thereof according to any one of (1″)-(7″), wherein the constant region of said heavy chain and the constant region of said light chain comprise a human antibody-derived sequence. (9″) The antibody or a Notch4 binding fragment thereof according to (8″), wherein the constant region of the heavy chain comprises the constant region of human IgG. (10″) The antibody or a Notch4 binding fragment thereof according to (9″), wherein said constant region of human IgG is the constant region of human IgG2. (11″) The antibody or a Notch4 binding fragment thereof according to (10″), wherein said constant region of human IgG2 has a mutation V234A and/or G237A. (12″) The antibody or a Notch4 binding fragment thereof according to (10″), wherein the lysine residue at the carboxy terminal of the constant region of said heavy chain is artificially removed. (13″) The antibody or a Notch4 binding fragment thereof according to (8″), wherein the constant region of said light chain comprises the constant region of human Igκ. (14″) A pharmaceutical composition comprising the anti-Notch4 antibody or a Notch4 binding fragment thereof according to any one of (1″)-(13″). (15″) The pharmaceutical composition according to (14″) which further comprises a pharmaceutically acceptable carrier. (16″) The pharmaceutical composition according to (15″) which is used for treatment of non-small cell lung cancer. (17″) The pharmaceutical composition according to (15″) which is used for treatment of thyroid cancer. (18″) The pharmaceutical composition according to (15″) which is used for treatment of prostate cancer. (19″) The pharmaceutical composition according to (15″) which is used for treatment of hepatocellular carcinoma.

An invention of any combination of one of more characteristics of the present invention listed above is also encompassed in the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship between the concentration of Antibody B and the relative luminescence (%) value.

FIGS. 2A and 2B show the antitumor effect and blood perfusion suppressive effect of Antibody B in a Calu6 xenograft model. FIG. 2A shows the change in relative tumor volume (RTV) for each group with tail vein administration of IgG at 10 mg/kg for Control or Antibody B at 1, 3, or 10 mg/kg in a Calu6 xenograft model (N=8, mean±standard error). FIG. 2B shows the result of determining the Hoechst fluorescence area for tumors sampled at the end of administration test (Day 8) (N=8 mean±standard error) (*P<0.05 vs Control IgG administration group (Dunnett test)).

FIG. 3 shows the antitumor effect due to the combination use of Antibody B and cisplatin in a Calu6 mouse subcutaneous transplantation model. The change in relative tumor volume (RTV) of the control (non-treated) group, Antibody B administration group (twice-a-week tail vein administration), cisplatin administration group (one-time tail vein administration), and Antibody B (twice-a-week tail vein administration)+cisplatin (one-time tail vein administration) combination group (N=4, mean±standard error) (*: P<0.05 vs control group (Student's t-test, Day 8, Day 24), #: P<0.05 cisplatin 10 mg/kg administration group vs Antibody B+cisplatin combination group (Student's t-test, Day 36)).

FIG. 4 shows the antitumor effect due to the combination use of Antibody B and lenvatinib mesylate in a FTC238 human thyroid cancer cell line xenograft model. The change in relative tumor volume (RTV) (N=5, mean±standard error) of the control (non-treated) group, Antibody B administration group (twice-a-week tail vein administration), lenvatinib mesylate administration group (once-a-day oral administration), and Antibody B (twice-a-week tail vein administration)+lenvatinib mesylate (once-a-day oral administration) combination group (*: P<0.05 vs control group (Student's t-test, Day 13), #: P<0.05 single agent administration group vs Antibody B+lenvatinib mesylate combination group (Student's t-test, Day 13)).

FIG. 5 shows the antitumor effect due to the combination use of Antibody B and paclitaxel in a DU145 human prostate cancer cell line xenograft model. The change in tumor volume (TV) of the control (non-treated) group, Antibody B administration group (twice-a-week tail vein administration), paclitaxel administration group (once-a-day 5-day tail vein administration), and Antibody B (twice-a-week tail vein administration)+paclitaxel (once-a-day 5-day tail vein administration) combination group (N=4, mean±standard error) (*: P<0.05 vs control group (Student's t-test, Day 57), #: P<0.05 single agent administration group vs Antibody B+paclitaxel combination group (Student's t-test, Day 57).

FIG. 6 shows the antitumor effect due to the combination use of Antibody B and lenvatinib mesylate in a human patient deribed hepatocellular carcinoma xenograft model. The change in tumor volume (TV) of the control (3 mam HCl) group, lenvatinib mesilate (10 mg/kg) gorop, sorafenib tosylate (30 mg/kg) group, and lenvatinib mesilate (10 mg/kg) plus Antibody B (0.5 mg/mouse) group (N=10, mean±standard error) (*: P<0.05 vs control group, unpaired t-test, Day 13).

FIG. 7 shows the relationship between the concentration of Antibody B and the relative luminescence (%) value. The graph shows the average value of three independent test results, and the error bar shows the standard deviation thereof.

FIGS. 8A-8D show the overlaid sensorgram of the interaction between Antibody B and human Notch NRR domains. (FIG. 8A) human Notch1-NRR-SEAP-His, (FIG. 8B) human Notch2-NRR-SEAP-His, (FIG. 8C) human Notch3-NRR-SEAP-His, and (FIG. 8D) human Notch4-NRR-SEAP-His.

DESCRIPTION OF EMBODIMENTS

An antibody herein may refer to an immunoglobulin molecule that can bind specifically to a target such as a sugar, a polynucleotide, a lipid, a polypeptide, and a protein via at least one antigen recognition site positioned on the variable region of the immunoglobulin molecule. An antibody may refer to a complete polyclonal or monoclonal antibody.

The antibody may be of any class such as IgG, IgA, or IgM (or a subclass thereof) etc. and is not limited to a particular class. An immunoglobulin is classified to different classes depending on the antibody amino acid sequence of the constant region of the heavy chain (sometimes referred to as the H chain). There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, some of which may be further classified into subclasses (isotypes) such as IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The constant regions of the heavy chain corresponding to the different classes of immunoglobulin are referred to as α, δ, ε, γ, and μ, respectively. Moreover, the types of the light chain (sometimes referred to as the L chain) of the antibody include λ and κ chains.

In one aspect, the anti-human Notch4 antibody of the present invention may be an IgG antibody, for example an IgG₁ antibody or an IgG₂ antibody etc. Moreover, in some cases, the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be in the form of a monomer, a dimer, or a multimer.

The antigen binding fragment of an antibody herein is not particularly limited, as long as it is a functional and structural fragment of said antibody and retains the binding ability to an antigen that can be bound by said antibody. Examples of the antigen binding fragment of an antibody include, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, single-chain (ScFv), variants thereof, a fusion protein comprising an antibody portion, other modified structures of an immunoglobulin molecule comprising the antigen recognition site, and the like.

The antigen binding fragment of an antibody can be obtained for example via proteolytic digestion of a complete antibody, or may be directly produced by a recombinant host cell (e.g. an eukaryote such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, or a prokaryote such as E. coli). For example, an F(ab′)₂ fragment may be formed by collecting Fab′-SH fragments directly from E. coli and subjecting them to chemical binding. F(ab′)₂ may also be formed by using a leucine zipper GCN4 which promotes the assembly of an F(ab′)₂ molecule. Moreover, an automatic synthesizer can be used when producing scFv with a chemical synthesis technology. An appropriate plasmid comprising a polynucleotide encoding scFv can be introduced to an appropriate host cell (e.g. an eukaryote such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, or a prokaryote such as E. coli) when producing scFv with a genetic recombination technology. The polynucleotide encoding the scFv of interest may be produced by a well-known manipulation such as ligation of polynucleotides. The scFv produced as a result may be isolated using a standard protein purification technology well-known in the art.

The variable region of an antibody may mean the variable region of the antibody light chain and/or the variable region of the antibody heavy chain, and the constant region of an antibody may mean the constant region of the antibody light chain and/or the constant region of the antibody heavy chain. The variable region of heavy and light chains each consists of four framework regions (FR) joined by three CDRs also known as hypervariable regions. The CDR in each chain is kept in the vicinity by a FR, and together with the CDR in the other chain contributes to the formation of the antigen binding site of the antibody. Technologies to determine CDRs include, but are not limited to, e.g. (1) an approach based on cross-species sequence variability (such as Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md.); and (2) an approach based on crystal structure research of antigen-antibody complexes (Al-lazikani et al., 1997 J. Molec. Biol. 273:927-948). These and other approaches may be employed in combination.

The term “binds specifically to” is a term well-known in the field to those skilled in the art, and methods for determining specific binding of an antibody etc. to an antigen or an epitope are also well-known. For example, it is understood that an antibody or an antigen binding fragment thereof that binds specifically to the epitope of Notch4 can bind to said Notch4 epitope with a higher affinity and binding activity, more rapidly, and/or, for a longer duration than to other epitope or non-epitope sites. However, an antibody or an antigen binding fragment thereof that binds specifically to a first target is not excluded from binding specifically to a second target.

A monoclonal antibody may mean an antibody that is obtained from a population of substantially uniform antibodies. In other words, individual antibodies contained in this population are identical except for a slight amount of naturally existing mutants that may be present. Monoclonal antibodies are directed to a single antigen site, and are very specific. Further, in contrast to a typical polyclonal antibody that targets different antigens or different epitopes, each monoclonal antibody targets a single epitope of the antigen. The modifier “monoclonal” indicates the property of an antibody that is obtained from a substantially uniform antibody population, and is not to be construed as being limited to requiring antibody production by a particular method.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be a chimeric antibody, a humanized antibody, a human antibody, a non-human mammal (such as mouse, rat, rabbit, cow, horse, and goat) antibody, or an antigen binding fragment thereof. A chimeric antibody is an antibody having e.g. the variable region of a non-human (such as mouse or rat) antibody introduced into the constant region of a human antibody, and may refer to e.g. an antibody wherein the variable region is derived from a non-human antibody and constant region is derived from a human antibody. A humanized antibody is an antibody having e.g. the hypervariable region (also referred to as complementarity determining region (CDR)) of a non-human antibody introduced into a human antibody, and may refer to e.g. an antibody wherein the CDR is derived from a non-human antibody and other antibody regions are derived from a human antibody. Note that in the present invention, the boundary between a chimeric antibody and a humanized antibody does not necessarily need to be clear, and an antibody may be in a state that may be called both a chimeric antibody and a humanized antibody.

Needless to say, the chimeric or humanized antibody exemplified above which has been appropriately modified (such as by modification of the antibody or partial substitution, addition, or deletion of the amino acid sequence of the antibody) while retaining the function of said antibody (or in order to add to or improve the function of said antibody) is also encompassed in the antibody of the present invention. More specifically, an antibody modified by the POTELLIGENT™ technology in order to increase the antibody-dependent cellular cytotoxicity ((ADCC) activity) of the antibody bound to the target, an antibody modified by the COMPLEGENT™ technology in order to increase the complement-dependent cytotoxicity ((CDC) activity) of the antibody, or an antibody modified by combination use of these technologies are also encompassed in the scope of the present invention. Moreover, an antibody having the lysine (Lys) located at the carboxy terminal (C-terminal) of the heavy chain deleted by an artificial method such as genetic modification in order to reduce the ununiformity of antibodies produced by antibody-producing cells is also encompassed in the scope of the present invention. Further, a bispecific antibody possessing the antibody binding site having the CDR sequence of the antibody of the present invention together with an antigen binding site that binds to a different antigen (Kontermann (2012), mAbs 4, 182-97) is also encompassed in the scope of the present invention.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be modified as desired. The modification of the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be a modification that changes (a) the three dimensional structure of the amino acid sequence at the modified region such as sheet or helix conformation; (b) the charge or hydrophobicity state of the molecule at the target site; or (c) the effect of modification on the side chain volume, or a modification where these changes are not clearly observed.

The modification of the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be achieved by e.g. substitution, deletion, and addition etc. of the configuring amino acid residues.

An amino acid herein is employed in its broadest meaning, and includes not only natural amino acids such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine (Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro), but also non-natural amino acids such as amino acid variants and derivatives. Those skilled in the art shall naturally recognize in light of this broad definition that examples of amino acids herein include L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; amino acids that are not materials configuring proteins in vivo such as norleucine, β-alanine, and ornithine; and chemically synthesized compounds having properties of amino acids well-known to those skilled in the art. Examples of a non-natural amino acid include α-methylamino acids (such as α-methylalanine), D-amino acids (such as D-aspartic acid and D-glutamic acid), histidine-like amino acids (such as 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids having excess methylene in the side chain (“homo” amino acids), and amino acids where the carboxylate functional group amino acid in the side chain is substituted with a sulfonate group (such as cysteic acid).

Naturally-occurring amino acid residues may be e.g. classified into the following groups based on common side chain properties:

(1) Hydrophobic: Met, Ala, Val, Leu, and Ile;

(2) Neutral hydrophilic: Cys, Ser, and Thr;

(3) Acidic: Asp and Glu;

(4) Basic: Asn, Gln, His, Lys, and Arg;

(5) Residues that influence chain orientation: Gly and Pro; and

(6) Aromatic: Trp, Tyr, and Phe.

A nonconservative substitution of the amino acid sequence configuring an antibody or an antigen binding fragment thereof may be performed by exchanging an amino acid that belongs to one of these groups with an amino acid that belongs to another group. A more conservative substitution may be performed by exchanging an amino acid that belongs to one of these groups with another amino acid that belongs to the same group. Similarly, deletion or substitution of the amino acid sequence may be appropriately performed.

A modification of the amino acid configuring an antibody or an antigen binding fragment thereof may be e.g. a post-translational modification such as glycosylation by a sugar, acetylation, or phosphorylation. The antibody may be glycosylated at a conserved position in its constant region. Glycosylation of an antibody is ordinarily either N-linked or O-linked. N-linked means linking of a sugar moiety to the side chain of an asparagine residue. Tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine (wherein X is any amino acid other than proline) are recognition sequences for enzymatically adding a sugar moiety to the asparagine side chain. A potential glycosylation site is present when one of these tripeptide sequences is present in an antibody or an antigen binding fragment thereof. O-linked glycosylation may be the linking of either N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (such as serine or threonine), and in some instances may be the linking to 5-hydroxy proline or 5-hydroxy lysine. The glycosylation condition (e.g. when glycosylation is performed with a biological means, the type of host cell or cell medium, pH, and the like) can be appropriately selected by those skilled in the art according to the purpose.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be further modified based on technical common sense well-known to those skilled in the art by other modification methods alone or in combination.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be produced by a method well-known to those skilled in the art. For example, the antibody may be produced with a hybridoma that produces the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof, or by integrating the gene encoding the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof into an expression vector and introducing said expression vector into e.g. E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, and the like.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be those that are isolated or purified according to methods well-known to those skilled in the art. Here, “isolated” or “purified” means that it is artificially isolated or purified from the natural state. When a molecule or a composition is naturally occurring, it is “isolated” or “purified” when it has changed or is removed from its original environment or both. Examples of an isolation or purification method include, but are not limited to, electrophoresis, molecular biological, immunological, or chromatographic means, specifically, ion exchange chromatography, hydrophobic chromatography, or reverse phase HPLC chromatography, or isoelectric focusing.

The method employed for measuring the binding property (such as binding affinity and cross-reactivity) of an antibody or an antigen binding fragment thereof to an antigen may be a method well-known in the field to those skilled in the art. For example, binding affinity may be measured with, but is not limited to, Biacore™ biosensor, KinExA biosensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (from IGEN), flow cytometry, fluorescence quenching, fluorescence transfer, yeast display, and/or immunostaining. The neutralizing activity of an antibody or an antigen binding fragment thereof against the binding of Notch4 to its ligand may be measured with, but is not limited to, Biacore™ biosensor, ELISA, and/or flow cytometry. The neutralizing activity an antibody or an antigen binding fragment thereof against signal transduction that is induced inside the human body due to the binding of Notch4 to its ligand, or against molecular expression response or functionality change of the cell may be measured with, but are not limited to, for example the following methods: (i) a reporter assay which detects variation in the expression of a molecule downstream of the Notch signal, (ii) Western Blot which detects Notch4 cleaving by TNF-α converting enzyme (TACE) or γ selectase, (iii) immune cell staining which detects nuclear import of Notch intracellular domain (NIC), and (iv) cell functionality evaluation which employs a normal cell such as a vascular endothelial cell or a cancer cell that expresses Notch4.

In one aspect, the present invention may be a pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof.

The pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof in an aqueous or dry preparation form may further comprise a pharmaceutically acceptable carrier, excipient, and/or a stabilizer. Examples of an acceptable carrier, excipient, or a stabilizer include saline; a buffer such as phosphoric acid, citric acid, and other organic acids; an antioxidant including ascorbic acid; a low molecular weight polypeptide; a protein (such as serum albumin, gelatin, or immunoglobulin); a hydrophilic polymer such as polyvinyl pyrrolidone; an amino acid; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; a chelator such as EDTA; sugar alcohols such as mannitol or sorbitol; a counter ion that forms a salt such as sodium; or a nonionic surfactant such as TWEEN™, PLURONICS™, or PEG.

The pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be encapsulated e.g. in a microcapsule, in a colloidal drug delivery system (such as a liposome, an albumin microsphere, a microemulsion, a nanoparticle, or a nanocapsule), or in a macroemulsion. When sustained release administration of the antibody is desired in a preparation having release property suitable for any disease that requires administration of the antibody, microcapsulation of the antibody may be intended. Examples of a sustained release matrix include a polyester, a hydrogel (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acids, a copolymer of L-glutamic acid and γ ethyl-L-glutamate, a nondegradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOT™ (an injectable microsphere composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxy butyric acid.

A preparation employed for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

The pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof has the potential of being useful for treatment of non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma. In other words, another aspect of the present invention may be a method for treating non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma comprising a step of administering to a subject a therapeutically effective amount the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof. Moreover, another aspect of the present invention may be a use of the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof for manufacturing a therapeutic drug for non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof used for treatment of non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma is preferably an antibody that recognizes the extracellular domain of Notch4. For example, the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be an antibody or an antigen binding fragment thereof that binds specifically to any site of positions 24-1447 in the amino acid sequence of human Notch4 shown in SEQ ID NO. 1. In the amino acid sequence of human Notch4 shown in SEQ ID NO. 1, positions 1-23 is the signal sequence, and positions 1448-2003 is the transmembrane domain and the intracellular domain.

The anti-Notch4 antibody of the present invention or an antigen binding fragment thereof can be used in a therapeutic method alone or in combination with other agents or compositions. For example, the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be administered at the same or different times with another anticancer agent. Such a combination therapy comprises combined administration (two or more agents are contained in the same or separate preparation) and separate administration (e.g. at the same time or continuously). When two or more agents are administered separately, the administration of the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be before or after the accompanying therapeutic method. The anticancer agent that may be used in combination with the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof may be e.g. an anticancer agent that is effective for treating non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma. Examples of such an anticancer agent can include, but are not limited to, cisplatin, lenvatinib, and paclitaxel. Examples of the pharmaceutical composition for such a combination therapy can include, but are not limited to, a pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof and cisplatin, a pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof and lenvatinib, and a pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof and paclitaxel.

The subject for administering the pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof is not limited, and the present invention can be employed for a mammal (such as a human, a pig, a cow, a monkey, a baboon, a dog, a cat, a rat, and a mouse). However, humans can be excluded from the subject when it is not preferred.

The administration method of the pharmaceutical composition comprising the anti-Notch4 antibody of the present invention or an antigen binding fragment thereof to a subject (such as administration route, dosage, frequency of administration per day, and administration timing) is not limited, and can be appropriately determined by those skilled in the art (such as a physician) according to the health state of the subject, the extent of disease, the type of agent used in combination, and the like.

It is recognized by those skilled in the art that as long as it is not technically contradicting, any one of more of any and all aspects described herein may be appropriately combined to carry out the present invention. Further, it is recognized by those skilled in the art that as long as it is not technically contradicting, it is preferred that any and all preferred or advantageous aspects described herein is appropriately combined to carry out the present invention.

All disclosures of the literatures cited herein should be deemed to be clearly cited herein by reference, and those skilled in the art will be able to cite and recognize the content of the disclosure related to these literatures as a part of the present specification according to the context herein without departing from the spirit and scope of the present invention.

The literatures cited herein are provided solely for the purpose of disclosing the related technology preceding the filing date of the present application, and is not to be construed as an admission that the present inventors do not hold the priority right to said disclosures for reasons of prior invention or any other reason. All description of these literatures are based on the information available to the present applicants, and do not configure the acknowledgement that these descriptions are correct.

The terms used herein are employed for describing particular embodiments and do not intend to limit the invention.

The term “comprise” as used herein, unless the content clearly indicates to be understood otherwise, intends the presence the described items (such as components, steps, elements, or numbers), and does not exclude the presence of other items (such as components, steps, elements, or numbers). The term “consist of” encompasses the aspects described with terms “consist of” and/or “consist essentially of.”

The term “neutralizing activity” as used herein means the activity to inhibit the binding of Notch4 to its ligand and/or the activity to inhibit signal transduction that is induced inside the human body by the binding of Notch4 to its ligand, or molecular expression response or functionality change of the cell.

Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meanings as those broadly recognized by those skilled in the art of the technology to which the present invention belongs. The terms used herein, unless explicitly defined otherwise, are to be construed as having meanings consistent with the meanings herein and in related technical fields, and shall not be construed as having idealized or excessively formal meanings.

Terms such as first and second are employed to express various elements, and it is recognized that these elements shall not be limited by these terms themselves. These terms are employed solely for the purpose of discriminating one element from another, and it is for example possible to describe a first element as a second element, and similarly to describe a second element as a first element without departing from the scope of the present invention.

The numeric values employed herein for indicating component content or numeric value range and the like, unless explicitly indicated, are to be understood as being modified by the term “about.” For example, unless explicitly indicated, “4° C.” is recognized as meaning “about 4° C.,” and those skilled in the art can naturally reasonably recognize the extent thereof according to technical common sense and the context of the present specification.

Unless clearly indicated to mean otherwise in context, when used in the specification and claims herein, it should be recognized that each aspect represented in singular form may also be a plural form as long as it is not technically contradicting, and vice versa.

The present invention will now be described in further detail with reference to Examples. However, the present invention can be embodied by various aspects, and shall not be construed as being limited to the Examples described herein. Those skilled in the art of related technical field will be able to carry out the present invention with various modifications, additions, deletions, substitution, and the like without altering the spirit or scope of the present invention.

EXAMPLES Example 1 Production of Anti-Human Notch4 Monoclonal Antibody

Production of Mouse Anti-Human Notch4 Monoclonal Antibody

In order to produce a monoclonal antibody against human Notch4 (Genbank Accession No. NP_004548.3) (SEQ ID NO. 1), Balb/c mice was immunized with three EGF repeats and the negative regulatory region (NRR) of human Notch4 (positions 1046-1445 of SEQ ID NO. 1) fused with secretory alkaline phosphatase (SEAP) and a histidine tag (hereinafter referred to as “human Notch4 3EGF-NRR-SEAP-His”).

Human Notch4 3EGF-NRR-SEAP-His protein was prepared by the following steps: First, an expression vector pcDNA3.1-human Notch4 3EGF-NRR-SEAP-His was constructed. The three EGF repeats and NRR of human Notch4 were amplified by PCR, and subcloned to the SfiI/NotI site of pcDNA3.1 (Invitrogen/LifeTechnologies) having a DNA sequence encoding an Igκ signal sequence, SEAP, and a histidine tag. Next, expression vector pcDNA3.1-human Notch4 3EGF-NRR-SEAP-His was transfected into HEK293 EBNA cells (Invitrogen/LifeTechnologies) by Trans IT-LT1 (TAKARA). After 6 days of incubation (5% CO2, 37° C.), the culture supernatant was collected. The human Notch4 3EGF-NRR-SEAP-His protein was purified with a Protino column (MACHEREY-NAGEL).

Twenty micrograms of said human Notch4 3EGF-NRR-SEAP-His protein was mixed with the same amount of GERBU adjuvant (GERBU Biotechnik GmbH) and subcutaneously injected into Balb/c mice footpad. Three additional injections were administered on Days 3, 7, and 10. Mice were sacrificed on the next day, and peripheral lymph nodes were collected. Half of each peripheral lymph nodes were transplanted into SCID mice. Lymph node cells were prepared from the remaining half of each lymph node, and fused to P3U1 myeloma cells at a proportion of 5:1 in the presence of GenomeONE-CF (Ishihara Sangyo Kaisha, Ltd.). Said fused cells were cultured in a 96-well plastic plate. After 7 days of incubation (5% CO2, 37° C.), the culture supernatant was collected.

Ten micrograms of human Notch4 3EGF-NRR-SEAP-His protein were intravenously administered to said lymph node transplantation SCID mice on the day of transplantation and 6 days after transplantation. Three days after the final immunization, peripheral lymph node cells were collected, fused as described above, and cultured.

Mouse monoclonal antibodies of 8 clones were obtained by the above steps. From these, the most preferred lead antibody (6-3-A6) was selected based on Notch4-specific signal inhibitory activity and binding activity to mouse Notch4 and human Notch4.

Sequence Analysis of Mouse Anti-Human Notch4 Monoclonal Antibody (6-3-A6)

The DNA sequence encoding the heavy and light chains of clone 6-3-A6 was amplified by 5′-RACE (5′-rapid amplification of cDNA ends). Whole RNA was prepared from said hybridoma with TRIZOL (Invitrogen/LifeTechnologies) and treated using DNase (QIAGEN, RNase free DNase set). Double-stranded cDNA was prepared from said whole RNA using cDNA synthesizing kit (TAKARA). The 5′ adaptor obtained by annealing of ad29S (ACATCACTCCGT (SEQ ID NO. 2)) and ad29AS (ACGGAGTGATGTCCGTCGACGTATCTCTGCGTTGATACTTCAGCGTAGCT (SEQ ID NO. 3)) was added to said cDNA. The cDNA obtained was amplified by 5′ forward primer (5′-PCR4 primer, AGCTACGCTGAAGTATCAACGCAGAG (SEQ ID NO. 4)) and 3′ reverse primer (GCCAGTGGATAGACTGATGG (SEQ ID NO. 5) was used for amplifying mouse IgG1 heavy chain and GATGGATACAGTTGGTGCAGC (SEQ ID NO. 6) was used for amplifying mouse Igκ light chain). Said amplified cDNA was inserted into pCR2.1 vector (Invitrogen/LifeTechnologies). The gene sequence was analyzed with ABI3130XL. The amino acid sequence encoded by the gene sequence identified by this analysis is shown in the following table.

TABLE 1 Amino Acid Sequence of Mouse Anti-human Notch4 Antibody (6-3-A6) Name Sequence Heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYGMS variable WVRQTPDKRLELVATINSNGGRTYYPDSVKGRFTI region SRDNAKNTLYLQMSSLKSEDTAMYYCARDQGFAYW (SEQ ID NO. 7) GQGTLVTVSA Light chain DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAW variable YQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTD region FTLTISNVQSEDLADYFCQQYSSYPWTFGGGTKLE (SEQ ID NO. 8) IK

TABLE 2 Nucleic Acid Sequence of Mouse Anti-human Notch4 Antibody (6-3-A6) Name Sequence Heavy chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGT variable GCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAG region CCTCTGGATTCACTTTCAGTAGCTATGGCATGTCT (SEQ ID NO. 9) TGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTT GGTCGCAACCATTAATAGTAATGGTGGTAGAACCT ATTATCCAGACAGTGTGAAGGGCCGATTCACCATC TCCAGAGACAATGCCAAGAACACCCTGTACCTGCA AATGAGCAGTCTGAAGTCTGAGGACACAGCCATGT ATTACTGTGCAAGAGACCAGGGTTTTGCTTACTGG GGCCAAGGGACTCTGGTCACTGTCTCTGCA Light chain GACATTGTGATGACCCAGTCTCACAAATTCATGTC variable CACATCAGTAGGAGACAGGGTCAGCATCACCTGCA region AGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGG (SEQ ID TATCAACAGAAACCAGGGCAATCTCCTAAACTACT NO. 10) GATTTACTGGGCATCCACCCGGCACACTGGAGTCC CTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATTAGCAATGTGCAGTCTGAAGA CTTGGCAGATTATTTCTGTCAGCAATATAGCAGCT ATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAA Preparation of Chimeric Anti-Human Notch4 Antibody and Humanized Anti-Human Notch4 Antibody

With overlapping extension PCR, the gene sequence of the heavy chain variable region of 6-3-A6 was bound to the gene sequence of the constant region of human IgG2 having mutations V234A and G237A as the heavy chain, and the gene sequence of the light chain variable region of 6-3-A6 was bound to the gene sequence of the constant region of human Igκ as the light chain to prepare a DNA sequence encoding a chimeric antibody. As used herein, “V234A” represents a mutation in which valine at position 234 is substituted with alanine and “G237A” represents a mutation in which glycine at position 237 is substituted with alanine. The sequence obtained as a result was inserted into expression vectors (pEE6.4 for heavy chain and pEE12.4 for light chain, Lonza). The amino acid and nucleotide sequences of the chimeric antibody are shown in the following tables.

TABLE 3 Amino Acid Sequence of Chimeric Anti-human Notch4 Antibody Sequence (The variable region is indicated in bold, and CDRs determined with Kabat definition method in the variable region Name are underlined.) Heavy  EVQLVESGGGLVQPGGSLKLSCAASGFTFS SYGMS WVRQTPD chain KRLELVA TINSNGGRTYYPDSVKG RFTISRDNAKNTLYLQMS (SEQ ID SLKSEDTAMYYCAR DQGFAY WGQGTLVTVSAASTKGPSVFPL NO. 11) APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Light DIVMTQSHKFMSTSVGDRVSITC KASQDVGTAVA WYQQKPGQ chain SPKLLIY WASTRHT GVPDRFTGSGSGTDFTLTISNVQSEDLA (SEQ ID DYFC QQYSSYPWT FGGGTKLEIKRTVAAPSVFIFPPSDEQLK NO. 12) SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

TABLE 4 Nucleic Acid Sequence of Chimeric Anti-human Notch4 Antibody Sequence (The variable region is indicated Name in bold.) Heavy GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCT chain GGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACT (SEQ ID TTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGAC NO. 13) AAGAGGCTGGAGTTGGTCGCAACCATTAATAGTAATGGTGGT AGAACCTATTATCCAGACAGTGTGAAGGGCCGATTCACCATC TCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGC AGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGA GACCAGGGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACT GTCTCTGCAGCTAGCACAAAAGGCCCCTCTGTCTTCCCTCTG GCTCCCTGCTCCCGCTCCACCTCCGAGTCCACTGCCGCTCTG GGCTGTCTGGTCAAGGATTACTTCCCTGAGCCAGTCACTGTG AGTTGGAACTCAGGCGCCCTGACCAGCGGAGTCCACACATTT CCCGCTGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAGT GTGGTCACCGTGCCTTCAAGCAATTTCGGGACTCAGACCTAT ACATGCAACGTGGACCATAAGCCATCTAATACTAAGGTCGAT AAAACCGTGGAGCGAAAATGCTGCGTGGAATGCCCACCTTGT CCTGCTCCACCAGCCGCTGCACCAAGCGTGTTCCTGTTTCCT CCAAAGCCCAAAGACACACTGATGATCAGCAGAACTCCTGAG GTCACCTGCGTGGTCGTGGACGTGTCCCACGAGGATCCCGAA GTCCAGTTTAACTGGTACGTGGATGGGGTCGAAGTGCATAAT GCAAAGACTAAACCTCGGGAGGAACAGTTCAACTCTACCTTT AGAGTCGTGAGTGTGCTGACAGTCGTGCACCAGGACTGGCTG AACGGAAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGCCTG CCCGCCCCTATCGAGAAAACAATTAGTAAGACTAAAGGCCAG CCAAGGGAACCCCAGGTGTACACACTGCCCCCTAGTCGCGAG GAAATGACAAAGAACCAGGTCTCACTGACTTGTCTGGTGAAA GGGTTCTATCCATCCGACATTGCCGTGGAGTGGGAATCTAAT GGACAGCCCGAAAACAATTACAAGACCACACCACCCATGCTG GACAGCGATGGATCCTTCTTTCTGTATTCAAAGCTGACCGTG GATAAAAGCCGGTGGCAGCAGGGCAATGTCTTTTCCTGCTCT GTGATGCACGAAGCCCTGCACAACCACTACACTCAGAAGTCC CTGTCCCTGTCTCCTGGCAAATGA Light GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCA chain GTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGAT (SEQ ID GTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAA NO. 14) TCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCA GATTATTTCTGTCAGCAATATAGCAGCTATCCGTGGACGTTC GGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTCGCCGCC CCCTCCGTGTTTATTTTTCCTCCATCTGACGAACAGCTGAAG AGTGGGACCGCCTCCGTGGTGTGCCTGCTGAACAATTTCTAC CCCCGGGAGGCCAAGGTGCAGTGGAAAGTCGACAACGCTCTG CAGTCTGGCAATAGTCAGGAGTCAGTGACTGAACAGGACAGC AAGGATTCCACCTATTCTCTGAGCTCCACCCTGACACTGAGC AAAGCAGATTACGAAAAGCACAAAGTCTATGCCTGCGAAGTG ACCCACCAGGGGCTGAGCAGTCCAGTGACCAAGTCCTTTAAC AGGGGAGAGTGTTGA

The antibody was humanized by transplanting mouse antibody 6-3-A6 CDR into the variable region of a human antibody. The amino acid sequence of mouse antibody 6-3-A6 was numbered according to the Kabat numbering system using Abysis software (licensed from UCL), and based on this numbering, said CDR was determined according to the Kabat definition or AbM definition method for identifying CDR. The amino acid and nucleotide sequences of 6-3-A6 CDR are shown in the following tables.

TABLE 5 Amino Acid Sequence of 6-3-A6 CDR Name Sequence Heavy chain CDR 1 SYGMS (Kabat definition) (SEQ ID NO. 15) Heavy chain CDR 1 GFTESSYGMS (AbM definition) (SEQ ID NO. 16) Heavy chain CDR 2 TINSNGGRTYYPDSVKG (Kabat definition) (SEQ ID NO. 17) Heavy chain CDR 2 TINSNGGRTY (AbM definition) (SEQ ID NO. 18) Heavy chain CDR 3 DQGFAY (SEQ ID NO. 19) Light chain CDR 1 KASQDVGTAVA (SEQ ID NO. 20) Light chain CDR 2 WASTRHT (SEQ ID NO. 21) Light chain CDR 3 QQYSSYPWT (SEQ ID NO. 22)

TABLE 6 Nucleic Acid Sequence of 6-3-A6 CDR Name Sequence Heavy chain CDR 1 AGCTATGGCATGTCT (Kabat definition) (SEQ ID NO. 23) Heavy chain CDR 1 GGATTCACTTTCAGTAGCTATGGCATGTCT (AbM definition) (SEQ ID NO. 24) Heavy chain CDR 2 ACCATTAATAGTAATGGTGGTAGAACCTAT (Kabat definition) TATCCAGACAGTGTGAAGGGC (SEQ ID NO. 25) Heavy chain CDR 2 ACCATTAATAGTAATGGTGGTAGAACCTAT (AbM definition) (SEQ ID NO. 26) Heavy chain CDR 3 GACCAGGGTTTTGCTTAC (SEQ ID NO. 27) Light chain CDR 1 AAGGCCAGTCAGGATGTGGGTACTGCTGT (SEQ ID NO. 28) AGCC Light chain CDR 2 TGGGCATCCACCCGGCACACT (SEQ ID NO. 29) Light chain CDR 3 CAGCAATATAGCAGCTATCCGTGGACG (SEQ ID NO. 30)

Based on the high homology to the framework region (FR) of 6-3-A6, FR of a human antibody, IGKV1-27*1 or IGKV3-15*1 and JK1 for the light chain, and IGHV3-64*01 and JH4 for the heavy chain were selected as the FR of the humanized antibody. Then, a 3D structure prediction model of mouse 6-3-A6 was employed to predict the amino acid in the FR that interacts with the amino acid of CDR, and transplanted together with CDR. The constant region of human IgG2 with mutations V234A and G237A and with or without a C-terminal lysine residue, as well as human Igκ were each employed as the constant region of heavy and light chains. HK1, HK2, and HK3 were designed as the heavy chain of the humanized antibody to which CDR determined by the Kabat definition method was transplanted, HA1 and HA2 were designed as the heavy chain of the humanized antibody to which CDR determined by the AbM definition method was transplanted, L1, L2, and L5 were designed as the light chain of the humanized antibody that employs IGKV1-27*1 and JK1, and L3, L4, and L6 were designed as the light chain of the humanized antibody that employs IGKV3-15*1 and JK1. The following tables show the amino acid and nucleotide sequences of the variable region of the humanized antibody, the constant region of human IgG2 with mutations V234A and G237A and with or without a C-terminal lysine residue, as well as human Igκ.

TABLE 7 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Heavy Chain (HK1) Sequence (CDRs determined with the Kabat Name definition method are underlined.) Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPG acid KGLEYVSTINSNGGRTYYPDSVKGRFTISRDNSKNTLYLQMG sequence SLRAEDMAVYYCARDQGFAYWGQGTLVTVSS (SEQ ID NO. 31) Nucleic GAGGTGCAGCTGGTCGAGAGCGGAGGGGGGCTGGTGCAGCCA acid GGAGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACC sequence TTCAGCTCCTACGGGATGTCTTGGGTGCGCCAGGCTCCAGGG (SEQ ID AAGGGACTGGAGTATGTCAGCACCATCAACTCCAATGGAGGC NO. 32) CGAACATACTATCCTGACTCCGTGAAGGGCCGGTTCACTATC TCTAGAGATAACAGTAAGAACACCCTGTACCTGCAGATGGGC AGCCTGAGAGCAGAAGACATGGCCGTCTACTATTGTGCAAGG GATCAGGGATTCGCATACTGGGGACAGGGAACTCTGGTGACC GTCTCAAGC

TABLE 8 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Heavy Chain (HK2) Sequence CDRs determined with the Kabat Name definition method are underlined.) Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPG acid KGLELVSTINSNGGRTYYPDSVKGRFTISRDNSKNTLYLQMG sequence SLRAEDMAVYYCARDQGFAYWGQGTLVTVSS (SEQ ID NO. 33) Nucleic GAAGTGCAGCTGGTCGAGAGCGGGGGAGGGCTGGTGCAGCCA acid GGAGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACC sequence TTCAGCTCCTACGGGATGTCTTGGGTGCGCCAGGCTCCAGGG (SEQ ID AAGGGACTGGAGCTGGTCAGCACCATCAACTCCAATGGAGGC NO. 34) CGAACATACTATCCTGACTCCGTGAAGGGCCGGTTCACTATC TCTAGAGATAACAGTAAGAACACCCTGTATCTGCAGATGGGC AGCCTGAGAGCAGAAGACATGGCCGTCTACTATTGTGCCCGA GATCAGGGGTTCGCTTATTGGGGACAGGGGACACTGGTGACC GTGAGCAGC

TABLE 9 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Heavy Chain (HK3) Sequence (CDRs determined with the Kabat Name definition method are underlined.) Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPG acid KGLELVATINSNGGRTYYPDSVKGRFTISRDNSKNTLYLQMG sequence VSLKAEDMAYYCARDQGFAYWGQGTLVTVSS (SEQ ID NO. 35) Nucleic GAAGTGCAGCTGGTCGAGAGTGGGGGAGGCCTGGTGCAGCCA acid GGAGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACC sequence TTCAGCTCCTACGGGATGTCCTGGGTGCGCCAGGCTCCAGGG (SEQ ID AAAGGACTGGAGCTGGTCGCCACCATCAACTCTAATGGAGGC NO. 36) CGAACATACTATCCTGACAGTGTGAAGGGCCGGTTCACTATT AGCAGAGATAACTCCAAAAATACCCTGTATCTGCAGATGGGC AGCCTGAAGGCAGAAGACATGGCCGTCTACTATTGTGCTCGG GATCAGGGGTTCGCCTATTGGGGGCAGGGGACTCTGGTCACT GTCTCTTCC

TABLE 10 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Heavy Chain (HA1) Sequence (CDRs determined with the AbM Name definition method are underlined.) Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPG acid KGLEYVSTINSNGGRTYYANSVKGRFTISRDNSKNTLYLQMG sequence SLRAEDMAVYYCARDQGFAYWGQGTLVTVSS (SEQ ID NO. 37) Nucleic GAAGTGCAGCTGGTCGAATCTGGGGGGGGACTGGTGCAGCCA acid GGAGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACC sequence TTCAGCTCCTACGGGATGTCTTGGGTGCGCCAGGCTCCTGGG (SEQ ID AAGGGACTGGAGTATGTCAGCACCATCAACTCCAATGGAGGC NO. 38) CGAACATACTATGCCAACTCCGTGAAGGGCCGGTTCACTATC TCTAGAGACAACAGTAAGAACACCCTGTACCTGCAGATGGGC AGCCTGAGAGCAGAAGATATGGCCGTCTACTATTGTGCTCGG GATCAGGGCTTTGCTTATTGGGGACAGGGGACACTGGTCACC GTCTCCTCC

TABLE 11 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Heavy Chain (HA2) Sequence (CDRs determined with the AbM Name definition method are underlined.) Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPG acid KGLELVSTINSNGGRTYYANSVKGRFTISRDNSKNTLYLQMG sequence SLRAEDMAVYYCARDQGFAYWGQGTLVTVSS (SEQ ID NO. 39) Nucleic GAGGTGCAGCTGGTCGAATCCGGGGGGGGGCTGGTGCAGCCA acid GGAGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACC sequence TTCAGCTCCTACGGGATGTCTTGGGTGCGCCAGGCTCCTGGG (SEQ ID AAGGGACTGGAGCTGGTCAGCACCATCAACTCCAATGGAGGC NO. 40) CGAACATACTATGCCAACTCCGTGAAGGGCCGGTTCACTATC TCTAGAGACAACAGTAAGAACACCCTGTATCTGCAGATGGGC AGCCTGAGAGCAGAAGATATGGCCGTCTACTATTGTGCTCGG GATCAGGGCTTCGCCTACTGGGGGCAGGGAACACTGGTCACC GTCTCCTCA

TABLE 12 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L1) Sequence (CDRs determined with the Kabat Name definition method are underlined.) Amino DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGK acid VPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDVA sequence TYYCQQYSSYPWTFGQGTKVEIK (SEQ ID NO. 41) Nucleic GACATTCAGATGACACAGAGCCCTTCATCTCTGAGTGCATCA acid GTGGGAGACAGGGTCACCATCACATGCAAAGCCAGCCAGGAT sequence GTGGGAACCGCAGTCGCTTGGTACCAGCAGAAGCCCGGGAAA (SEQ ID GTGCCTAAGCTGCTGATCTACTGGGCTAGTACACGGCACACT NO. 42) GGCGTCCCATCCAGATTCAGCGGCTCCGGGTCTGGAACCGAC TTTACTCTGACCATCAGCTCCCTGCAGCCCGAGGATGTGGCC ACATACTATTGCCAGCAGTATTCATCTTATCCTTGGACCTTC GGACAGGGAACAAAAGTGGAAATCAAA

TABLE 13 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L2) Sequence (CDRs determined with the Kbat Name definition method are underlined.) Amino acid DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKVPKL sequence LIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYS (SEQ ID NO. 43) SYPWTFGQGTKVEIK Nucleic acid GATATTCAGATGACTCAGAGCCCCTCCTCTCTGAGTGCATCAGTGG sequence GAGACAGGGTCACCATCACATGCAAAGCCAGCCAGGATGTGGGAAC (SEQ ID NO. 44) CGCAGTCGCTTGGTACCAGCAGAAGCCCGGGAAAGTGCCTAAGCTG CTGATCTACTGGGCTAGTACACGGCACACTGGCGTCCCATCCAGAT TCAGCGGCTCCGGGTCTGGAACCGACTTTACTCTGACCATCAGCTC CCTGCAGCCCGAGGATGTGGCCACATACTTCTGCCAGCAGTATTCA TCCTATCCTTGGACCTTCGGACAGGGAACTAAAGTGGAGATTAAG

TABLE 14 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L3) Sequence (CDRs determined with the Rabat Name definition method are underlined.) Amino acid EIVMTQSPATLSVSPGERATLSCKASQDVGTAVAWYQQKPGQAPRL sequence LIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYS (SEQ ID NO. 45) SYPWTFGQGTKVEIK Nucleic acid GAAATTGTGATGACCCAGTCTCCCGCCACACTGTCTGTGAGTCCAG sequence GAGAGAGGGCAACTCTGTCTTGCAAGGCCAGTCAGGACGTGGGAAC (SEQ ID NO. 46) CGCAGTCGCTTGGTACCAGCAGAAACCCGGGCAGGCTCCTCGGCTG CTGATCTATTGGGCATCCACTCGGCACACCGGCATTCCCGCCAGAT TCTCAGGCAGCGGGTCCGGAACAGAGTTTACCCTGACAATCAGCTC CCTGCAGAGCGAAGATTTCGCTGTCTACTATTGCCAGCAGTATTCT AGTTATCCTTGGACATTCGGCCAGGGAACAAAAGTGGAAATCAAA

TABLE 15 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L4) Sequence (CDRs determined with the Kabat Name definition method are underlined.) Amino acid EIVMTQSPATLSVSPGERATLSCKASQDVGTAVAWYQQKPGQAPRL sequence LIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYS (SEQ ID NO. 47) SYPWTFGQGTKVEIK Nucleic acid GAAATCGTGATGACCCAGAGCCCCGCAACACTGTCTGTGAGTCCAG sequence GAGAGAGGGCAACTCTGTCTTGCAAGGCCAGTCAGGACGTGGGAAC (SEQ ID NO. 48) CGCAGTCGCTTGGTACCAGCAGAAACCCGGGCAGGCTCCTCGGCTG CTGATCTATTGGGCATCCACTCGGCACACCGGCATTCCCGCCAGAT TCTCAGGCAGCGGGTCCGGAACAGAGTTTACCCTGACAATCAGCTC CCTGCAGAGCGAAGATTTCGCTGTCTACTTTTGCCAGCAGTATTCA TCCTATCCTTGGACCTTCGGACAGGGAACAAAAGTGGAAATCAAA

TABLE 16 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L5) Sequence CDRs determined with the Kbat Name definition method are underlined.) Amino acid DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKL sequence LIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYS (SEQ ID NO. 49) SYPWTFGQGTKVEIK Nucleic acid GATATCCAGATGACCCAGTCCCCAAGCTCCCTGTCCGCATCTGTGG sequence GCGACCGGGTCACCATTACATGTAAAGCCAGTCAGGATGTGGGAAC (SEQ ID NO. 50) AGCCGTCGCTTGGTACCAGCAGAAGCCCGGCAAATCTCCTAAGCTG CTGATCTATTGGGCTTCCACACGGCACACTGGCGTGCCCTCTAGAT TCAGTGGCTCAGGGAGCGGAACAGACTTTACTCTGACCATTTCTAG TCTGCAGCCAGAGGATGTGGCAACTTACTTCTGCCAGCAGTACTCA AGCTATCCCTGGACCTTTGGCCAGGGGACAAAAGTCGAAATCAAG

TABLE 17 Amino Acid and Nucleic Acid Sequences of the Variable Region of Humanized Anti-human Notch4 Antibody Light Chain (L6) Sequence (CDRs determined with the Kabat Name definition method are underlined.) Amino acid EIVMTQSPATLSVSPGERATLSCKASQDVGTAVAWYQQKPGQSPRL sequence LIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYS (SEQ ID NO. 51) SYPWTFGQGTKVEIK Nucleic acid GAAATCGTGATGACCCAGAGCCCCGCAACACTGTCTGTGAGTCCAG sequence GAGAGAGGGCAACTCTGTCTTGCAAGGCCAGTCAGGACGTGGGAAC (SEQ ID NO. 52) CGCAGTCGCTTGGTACCAGCAGAAACCCGGGCAGTCTCCTCGGCTG CTGATCTATTGGGCATCCACTCGGCACACCGGCATTCCCGCCAGAT TCTCAGGCAGCGGGTCCGGAACAGAGTTTACCCTGACAATCAGCTC CCTGCAGAGCGAAGATTTCGCTGTCTACTTTTGCCAGCAGTATTCA TCCTATCCTTGGACCTTCGGACAGGGAACAAAAGTGGAAATCAAA

TABLE 18 Amino Acid and Nucleic Acid Sequences of the Constant Region of Human IgG2 with Mutations V234A and  G237A and C-terminal Lysine Name Sequence Amino acid ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL sequence TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT (SEQ ID NO. 53) KVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Nucleic acid GCTAGCACAAAAGGCCCCTCTGTCTTCCCTCTGGCTCCCTGCTCCC sequence GCTCCACCTCCGAGTCCACTGCCGCTCTGGGCTGTCTGGTCAAGGA (SEQ ID NO. 54) TTACTTCCCTGAGCCAGTCACTGTGAGTTGGAACTCAGGCGCCCTG ACCAGCGGAGTCCACACATTTCCCGCTGTGCTGCAGAGCTCCGGCC TGTACTCCCTGTCTAGTGTGGTCACCGTGCCTTCAAGCAATTTCGG GACTCAGACCTATACATGCAACGTGGACCATAAGCCATCTAATACT AAGGTCGATAAAACCGTGGAGCGAAAATGCTGCGTGGAATGCCCAC CTTGTCCTGCTCCACCAGCCGCTGCACCAAGCGTGTTCCTGTTTCC TCCAAAGCCCAAAGACACACTGATGATCAGCAGAACTCCTGAGGTC ACCTGCGTGGTCGTGGACGTGTCCCACGAGGATCCCGAAGTCCAGT TTAACTGGTACGTGGATGGGGTCGAAGTGCATAATGCAAAGACTAA ACCTCGGGAGGAACAGTTCAACTCTACCTTTAGAGTCGTGAGTGTG CTGACAGTCGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGT GCAAAGTGTCTAATAAGGGCCTGCCCGCCCCTATCGAGAAAACAAT TAGTAAGACTAAAGGCCAGCCAAGGGAACCCCAGGTGTACACACTG CCCCCTAGTCGCGAGGAAATGACAAAGAACCAGGTCTCACTGACTT GTCTGGTGAAAGGGTTCTATCCATCCGACATTGCCGTGGAGTGGGA ATCTAATGGACAGCCCGAAAACAATTACAAGACCACACCACCCATG CTGGACAGCGATGGATCCTTCTTTCTGTATTCAAAGCTGACCGTGG ATAAAAGCCGGTGGCAGCAGGGCAATGTCTTTTCCTGCTCTGTGAT GCACGAAGCCCTGCACAACCACTACACTCAGAAGTCCCTGTCCCTG TCTCCTGGCAAATGA

TABLE 19 Amino Acid and Nucleic Acid Sequences of the Constant Region of Human IgG2 With Mutations V234A and G237A and Without C-terminal Lysine Name Sequence Amino acid ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL sequence TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT (SEQ ID NO. 55) KVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG Nucleic acid GCTAGCACAAAAGGCCCCTCTGTCTTCCCTCTGGCTCCCTGCTCCC sequence GCTCCACCTCCGAGTCCACTGCCGCTCTGGGCTGTCTGGTCAAGGA (SEQ ID NO. 56) TTACTTCCCTGAGCCAGTCACTGTGAGTTGGAACTCAGGCGCCCTG ACCAGCGGAGTCCACACATTTCCCGCTGTGCTGCAGAGCTCCGGCC TGTACTCCCTGTCTAGTGTGGTCACCGTGCCTTCAAGCAATTTCGG GACTCAGACCTATACATGCAACGTGGACCATAAGCCATCTAATACT AAGGTCGATAAAACCGTGGAGCGAAAATGCTGCGTGGAATGCCCAC CTTGTCCTGCTCCACCAGCCGCTGCACCAAGCGTGTTCCTGTTTCC TCCAAAGCCCAAAGACACACTGATGATCAGCAGAACTCCTGAGGTC ACCTGCGTGGTCGTGGACGTGTCCCACGAGGATCCCGAAGTCCAGT TTAACTGGTACGTGGATGGGGTCGAAGTGCATAATGCAAAGACTAA ACCTCGGGAGGAACAGTTCAACTCTACCTTTAGAGTCGTGAGTGTG CTGACAGTCGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGT GCAAAGTGTCTAATAAGGGCCTGCCCGCCCCTATCGAGAAAACAAT TAGTAAGACTAAAGGCCAGCCAAGGGAACCCCAGGTGTACACACTG CCCCCTAGTCGCGAGGAAATGACAAAGAACCAGGTCTCACTGACTT GTCTGGTGAAAGGGTTCTATCCATCCGACATTGCCGTGGAGTGGGA ATCTAATGGACAGCCCGAAAACAATTACAAGACCACACCACCCATG CTGGACAGCGATGGATCCTTCTTTCTGTATTCAAAGCTGACCGTGG ATAAAAGCCGGTGGCAGCAGGGCAATGTCTTTTCCTGCTCTGTGAT GCACGAAGCCCTGCACAACCACTACACTCAGAAGTCCCTGTCCCTG TCTCCTGGCTGA

TABLE 20 Amino Acid and Nucleic Acid Sequences of the Constant Region of Human Igκ Name Sequence Amino acid RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA sequence LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ (SEQ ID NO. 57) GLSSPVTKSFNRGEC Nucleic acid CGTACGGTCGCCGCCCCCTCCGTGTTTATTTTTCCTCCATCTGACG sequence AACAGCTGAAGAGTGGGACCGCCTCCGTGGTGTGCCTGCTGAACAA (SEQ ID NO. 58) TTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAAGTCGACAACGCT CTGCAGTCTGGCAATAGTCAGGAGTCAGTGACTGAACAGGACAGCA AGGATTCCACCTATTCTCTGAGCTCCACCCTGACACTGAGCAAAGC AGATTACGAAAAGCACAAAGTCTATGCCTGCGAAGTGACCCACCAG GGGCTGAGCAGTCCAGTGACCAAGTCCTTTAACAGGGGAGAGTGTT GA

The gene sequences of the variable region of these humanized antibodies were synthesized by GenScript USA Inc., and inserted into the constant region of human IgG2 with or without a C-terminal lysine or pcDNA3.3 (Invitrogen) comprising the DNA sequence encoding the constant region of human Igκ. Said expression vectors were transfected into FreeStyle 293-F cells (Invitrogen) using FreeStyle 293 expression system (Invitrogen) in order to produce the antibodies. The supernatant was collected and purified with Protein A (GE Healthcare).

The full length (variable region+constant region) gene sequences of the humanized antibodies were similarly optimized, fully synthesized by GenScript USA Inc., and the heavy chain was inserted into pEE6.4 and the light chain into pEE12.4 (Lonza). These expression vectors were employed as above in order to produce the antibodies. The optimized nucleotide sequences of the humanized antibodies are shown in the following tables.

TABLE 21 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Heavy Chain (HK2 Variable Region + Human IgG2 Constant Region with Mutations V234A and G237A and Without C-terminal Lysine) Name Sequence Nucleic acid GAAGTGCAGCTGGTCGAATCTGGGGGGGGTCTGGTGCAGCCAGGCG sequence GATCCCTGAGACTGAGCTGCGCCGCTTCTGGGTTCACATTTTCCAG (SEQ ID NO. 59) CTACGGCATGTCCTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAG CTGGTGAGTACAATCAACTCAAATGGGGGTCGAACTTACTATCCCG ACTCCGTGAAGGGCAGGTTCACTATTTCCCGGGATAACAGCAAAAA TACCCTGTACCTGCAGATGGGGTCCCTGCGAGCTGAAGACATGGCA GTGTACTATTGTGCCCGTGATCAGGGTTTCGCTTATTGGGGGCAGG GTACTCTGGTCACCGTGTCTAGTGCTTCTACCAAGGGACCATCCGT GTTCCCACTGGCACCATGCTCCCGGAGCACATCTGAGAGTACTGCA GCCCTGGGCTGTCTGGTGAAGGACTATTTCCCTGAACCAGTCACAG TGAGCTGGAACTCTGGCGCACTGACAAGCGGAGTCCACACTTTTCC TGCCGTGCTGCAGTCATCCGGCCTGTACTCTCTGAGCTCTGTGGTC ACTGTCCCCAGTTCAAATTTCGGAACTCAGACCTATACATGCAACG TGGACCATAAGCCTAGCAATACCAAGGTCGATAAAACAGTGGAGCG TAAATGCTGCGTGGAATGCCCACCTTGTCCAGCACCACCAGCTGCA GCCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCAAAAGACACCCTGA TGATCTCTAGAACCCCCGAGGTCACATGCGTGGTCGTGGACGTGAG TCACGAGGATCCTGAAGTCCAGTTTAACTGGTACGTGGATGGCGTC GAAGTGCATAATGCCAAGACAAAACCAAGAGAGGAACAGTTCAACT CAACCTTTCGCGTCGTGTCCGTGCTGACAGTCGTGCACCAGGATTG GCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCCAATAAGGGACTG CCCGCTCCTATCGAGAAAACTATTTCCAAGACCAAAGGACAGCCTA GGGAACCACAGGTGTACACTCTGCCCCCTTCCCGGGAGGAAATGAC TAAGAACCAGGTCAGCCTGACCTGTCTGGTGAAAGGGTTCTATCCT AGTGACATTGCCGTGGAGTGGGAATCAAATGGTCAGCCAGAGAACA ATTACAAGACCACACCACCCATGCTGGACAGTGATGGCTCATTCTT TCTGTATAGCAAGCTGACCGTCGATAAATCTAGGTGGCAGCAGGGA AACGTGTTCTCCTGCTCCGTGATGCACGAAGCACTGCACAACCATT ACACCCAGAAATCCCTGAGCCTGTCCCCCGGCTGA

TABLE 22 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Heavy Chain (HK3 Variable Region + Human IgG2 Constant Region with Mutations V234A and G237A and Without C-terminal Lysine) Name Sequence Nucleic acid GAGGTGCAGCTGGTCGAGTCCGGGGGGGGTCTGGTGCAGCCAGGAG sequence GATCCCTGAGGCTGAGCTGCGCCGCTTCTGGGTTCACATTTTCCAG (SEQ ID NO. 60) CTACGGCATGTCCTGGGTCCGGCAGGCACCAGGCAAGGGACTGGAG CTGGTGGCCACAATCAACAGTAATGGGGGTAGAACTTACTATCCCG ACTCAGTGAAGGGCAGGTTCACTATTAGTCGGGATAACTCAAAAAA TACCCTGTACCTGCAGATGGGGTCCCTGAAGGCTGAAGACATGGCA GTGTACTATTGTGCCCGCGATCAGGGTTTCGCTTATTGGGGGCAGG GTACTCTGGTCACCGTGTCTAGTGCCTCCACCAAAGGGCCCAGCGT GTTTCCACTGGCTCCCTGCTCCCGAAGCACATCTGAGAGTACTGCA GCCCTGGGCTGTCTGGTGAAGGACTATTTCCCTGAACCAGTCACAG TGAGCTGGAACTCTGGCGCTCTGACATCTGGAGTCCACACTTTTCC TGCAGTGCTGCAGTCATCCGGCCTGTACTCCCTGAGCTCTGTGGTC ACTGTCCCCAGTTCAAATTTCGGAACTCAGACCTATACATGCAACG TGGACCATAAACCTAGCAATACCAAGGTCGATAAAACAGTGGAGCG GAAGTGCTGTGTGGAATGCCCACCTTGTCCAGCTCCACCAGCTGCA GCCCCTTCTGTGTTCCTGTTTCCTCCAAAGCCAAAAGACACCCTGA TGATCAGCAGGACCCCCGAGGTCACATGTGTGGTCGTGGACGTGTC TCACGAGGATCCTGAAGTCCAGTTTAACTGGTACGTGGATGGCGTC GAAGTGCATAATGCAAAGACAAAACCAAGAGAGGAACAGTTCAACT CTACCTTTCGCGTCGTGAGTGTGCTGACAGTCGTGCACCAGGATTG GCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCCAATAAGGGACTG CCCGCCCCTATCGAGAAAACTATTAGCAAGACCAAAGGACAGCCTC GAGAACCACAGGTGTACACTCTGCCCCCTAGTCGTGAGGAAATGAC TAAGAACCAGGTCTCCCTGACCTGTCTGGTGAAAGGGTTCTATCCT AGCGACATTGCCGTGGAGTGGGAATCTAATGGTCAGCCAGAGAACA ATTACAAGACCACACCACCCATGCTGGACAGTGATGGCTCATTCTT TCTGTATTCAAAGCTGACCGTCGATAAATCCAGGTGGCAGCAGGGA AATGTGTTTTCATGCTCCGTGATGCACGAAGCCCTGCACAACCATT ACACCCAGAAGAGCCTGTCCCTGAGCCCCGGCTGA

TABLE 23 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Heavy Chain (HA2 Variable Region + Human IgG2 Constant Region with Mutations V234A and G237A and Without C-terminal Lysine) Name Sequence Nucleic acid GAAGTGCAGCTGGTCGAGTCTGGGGGGGGGCTGGTGCAGCCTGGCG sequence GATCCCTGAGACTGAGCTGCGCCGCTTCTGGGTTCACATTTTCCAG (SEQ ID NO. 61) CTACGGCATGTCCTGGGTCCGCCAGGCACCAGGCAAGGGACTGGAG CTGGTGAGTACAATCAACTCAAATGGGGGTCGAACTTACTATGCTA ACTCCGTGAAGGGCAGGTTCACTATTTCCCGGGACAACAGCAAAAA TACCCTGTACCTGCAGATGGGGTCCCTGCGAGCTGAAGACATGGCA GTGTACTATTGTGCCCGTGATCAGGGTTTCGCTTATTGGGGGCAGG GTACTCTGGTCACCGTGTCTAGTGCTTCTACCAAGGGGCCCAGTGT GTTTCCACTGGCACCCTGCTCCCGGAGCACATCTGAGAGTACTGCA GCCCTGGGCTGTCTGGTGAAGGATTATTTCCCTGAACCAGTCACAG TGAGCTGGAACTCTGGCGCACTGACAAGCGGAGTCCACACTTTTCC TGCCGTGCTGCAGTCATCCGGCCTGTACTCTCTGAGCTCTGTGGTC ACTGTCCCCAGTTCAAATTTCGGAACTCAGACCTATACATGCAACG TGGACCATAAGCCTAGCAATACCAAGGTCGATAAAACAGTGGAGCG TAAATGCTGTGTGGAATGCCCACCTTGTCCAGCTCCACCAGCTGCA GCCCCTTCTGTGTTCCTGTTTCCTCCAAAGCCAAAAGACACCCTGA TGATCTCTAGAACCCCCGAGGTCACATGTGTGGTCGTGGACGTCAG TCACGAGGATCCAGAAGTCCAGTTTAACTGGTACGTGGATGGCGTC GAAGTGCATAATGCAAAGACAAAACCCAGAGAGGAACAGTTCAACT CAACCTTTCGCGTCGTGTCCGTGCTGACAGTCGTGCACCAGGACTG GCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCCAATAAGGGACTG CCCGCCCCTATCGAGAAAACTATTTCCAAGACCAAAGGACAGCCTA GGGAACCACAGGTGTACACTCTGCCCCCTTCCCGGGAGGAAATGAC TAAGAACCAGGTCAGCCTGACCTGTCTGGTGAAAGGGTTCTATCCT AGTGACATTGCCGTGGAGTGGGAATCAAATGGTCAGCCAGAGAACA ATTACAAGACCACACCACCCATGCTGGACAGTGATGGCTCATTCTT TCTGTATAGCAAGCTGACCGTCGATAAATCTAGGTGGCAGCAGGGA AATGTGTTTTCATGCTCCGTGATGCACGAAGCCCTGCACAACCACT ACACACAGAAAAGCCTGAGCCTGAGCCCCGGCTGA

TABLE 24 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Light Chain (L3 Variable Region + Human Igκ Constant Region) Name Sequence Nucleic acid GAAATCGTGATGACTCAGTCCCCCGCTACACTGAGCGTGTCTCCCG sequence GAGAGAGAGCTACTCTGTCTTGCAAGGCAAGTCAGGACGTGGGAAC (SEQ ID NO. 62) TGCAGTCGCCTGGTACCAGCAGAAACCAGGACAGGCACCACGACTG CTGATCTATTGGGCTAGTACAAGGCACACTGGCATTCCTGCCCGGT TCAGTGGCTCAGGATCCGGGACAGAGTTTACCCTGACAATCTCCAG CCTGCAGTCCGAAGATTTCGCTGTGTACTATTGCCAGCAGTACTCT AGTTATCCTTGGACCTTTGGTCAGGGCACAAAGGTCGAGATCAAAC GAACCGTGGCCGCTCCAAGCGTCTTCATTTTTCCCCCTTCTGACGA ACAGCTGAAGTCAGGTACAGCCTCCGTGGTCTGTCTGCTGAACAAT TTCTACCCAAGGGAGGCAAAGGTGCAGTGGAAAGTCGATAACGCCC TGCAGAGCGGCAATTCTCAGGAGAGTGTGACTGAACAGGACTCAAA GGATTCCACCTATAGCCTGTCATCCACTCTGACCCTGAGCAAAGCT GACTACGAAAAGCATAAAGTGTATGCATGTGAAGTCACACACCAGG GTCTGAGTTCTCCAGTCACCAAATCTTTTAATAGAGGCGAGTGCTG A

TABLE 25 Optimized Nucleic Acid Sequence of Humanized Anti-human  Notch4 Antibody Light Chain (L4 Variable Region + Human Igκ Constant Region) Name Sequence Nucleic acid GAAATCGTGATGACCCAGTCTCCTGCTACACTGAGCGTGTCTCCCG sequence GAGAGAGAGCTACTCTGTCTTGCAAGGCAAGTCAGGACGTGGGAAC (SEQ ID NO. 63) TGCAGTCGCCTGGTACCAGCAGAAACCAGGACAGGCACCACGACTG CTGATCTATTGGGCTAGTACAAGGCACACTGGCATTCCTGCCCGGT TCAGTGGCTCAGGATCCGGGACAGAGTTTACCCTGACAATCTCCAG CCTGCAGTCCGAAGATTTCGCTGTGTACTTTTGCCAGCAGTACTCT AGTTATCCTTGGACCTTCGGTCAGGGCACAAAGGTCGAGATCAAAC GAACCGTGGCCGCTCCAAGCGTCTTCATTTTTCCCCCTTCTGACGA ACAGCTGAAGTCAGGTACAGCCTCCGTGGTCTGTCTGCTGAACAAT TTTTACCCAAGGGAGGCAAAGGTGCAGTGGAAAGTCGATAACGCCC TGCAGAGCGGCAATTCTCAGGAGAGTGTGACTGAACAGGACTCAAA GGATTCCACCTATAGCCTGTCATCCACTCTGACCCTGAGCAAAGCT GACTACGAAAAGCATAAAGTGTATGCATGTGAAGTCACACACCAGG GTCTGTCCAGTCCAGTCACCAAATCCTTTAATCGGGGAGAGTGCTG A

TABLE 26 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Light Chain (L5 Variable Region + Human Igκ Constant Region) Name Sequence Nucleic acid GATATTCAGATGACCCAGTCTCCTTCCAGCCTGTCTGCAAGTGTGG sequence GAGACAGGGTCACCATCACATGCAAAGCCTCCCAGGATGTGGGAAC (SEQ ID NO. 64) CGCAGTCGCCTGGTACCAGCAGAAGCCAGGGAAAAGCCCCAAGCTG CTGATCTACTGGGCTTCTACCAGGCACACAGGCGTGCCAAGTCGGT TCTCAGGCTCCGGAAGCGGGACCGACTTTACTCTGACCATCTCCAG CCTGCAGCCTGAGGATGTGGCAACATACTTCTGCCAGCAGTACTCT AGTTATCCATGGACTTTTGGTCAGGGCACCAAAGTCGAGATCAAGA GAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTAGCGACGA ACAGCTGAAGAGTGGTACAGCCTCAGTGGTCTGTCTGCTGAACAAT TTCTACCCTAGGGAGGCTAAAGTGCAGTGGAAGGTCGATAACGCAC TGCAGTCTGGCAATAGTCAGGAGTCAGTGACAGAACAGGACTCCAA AGATAGCACTTATTCTCTGTCATCCACACTGACTCTGTCTAAGGCC GACTACGAAAAGCATAAAGTGTATGCTTGTGAGGTCACACACCAGG GTCTGAGCAGTCCAGTCACCAAGAGCTTTAACCGAGGAGAGTGCTG A

TABLE 27 Optimized Nucleic Acid Sequence of Humanized Anti-human Notch4 Antibody Light Chain (L6 Variable Region + Human Igκ Constant Region) Name Sequence Nucleic acid GAAATCGTGATGACCCAGTCTCCTGCTACACTGAGCGTGTCTCCCG sequence GAGAGAGAGCTACTCTGTCTTGCAAGGCAAGTCAGGACGTGGGAAC (SEQ ID NO. 65) TGCAGTCGCCTGGTACCAGCAGAAACCAGGGCAGAGTCCCCGCCTG CTGATCTATTGGGCCTCCACAAGGCACACTGGCATTCCTGCTCGGT TCAGTGGCTCAGGATCCGGGACAGAGTTTACCCTGACAATCTCCAG CCTGCAGAGCGAAGATTTCGCCGTGTACTTTTGCCAGCAGTACTCT AGTTATCCTTGGACCTTCGGTCAGGGCACAAAGGTCGAGATCAAAC GAACCGTGGCCGCTCCAAGCGTCTTCATTTTTCCCCCTTCTGACGA ACAGCTGAAGTCAGGTACAGCTTCCGTGGTCTGTCTGCTGAACAAT TTTTACCCAAGGGAGGCAAAGGTGCAGTGGAAAGTCGATAACGCCC TGCAGAGCGGCAATTCTCAGGAGAGTGTGACTGAACAGGACTCAAA GGATTCCACCTATAGCCTGTCATCCACTCTGACCCTGTCTAAAGCT GACTACGAAAAGCATAAAGTGTATGCATGTGAAGTCACCCACCAGG GGCTGAGTAGTCCAGTCACCAAGAGTTTTAATCGGGGCGAGTGTTG A

In the following Examples, experiments were carried out with antibodies comprising the amino acid sequence of CDR of antibody 6-3-A6 determined in Example 1.

For convenience, the specific antibodies employed in the following Example will be referred to as “Antibody A,” “Antibody B,” “Antibody C,” “Antibody D,” “Antibody E,” “Antibody F,” and “Antibody G.”

In Antibody A, the heavy chain variable region comprises the heavy chain variable region of HK2 described in Example 1 and the light chain variable region comprises the light chain variable region of L3 described in Example 1.

In Antibody B, the heavy chain variable region comprises the heavy chain variable region of HK3 described in Example 1 and the light chain variable region comprises the light chain variable region of L3 described in Example 1.

In Antibody C, the heavy chain variable region comprises the heavy chain variable region of HK2 described in Example 1 and the light chain variable region comprises the light chain variable region of L4 described in Example 1.

In Antibody D, the heavy chain variable region comprises the heavy chain variable region of HK3 described in Example 1 and the light chain variable region comprises the light chain variable region of L5 described in Example 1.

In Antibody E, the heavy chain variable region comprises the heavy chain variable region of HK2 described in Example 1 and the light chain variable region comprises the light chain variable region of L6 described in Example 1.

In Antibody F, the heavy chain variable region comprises the heavy chain variable region of HA2 described in Example 1 and the light chain variable region comprises the light chain variable region of L3 described in Example 1.

In Antibody G, the heavy chain variable region comprises the heavy chain variable region of HK3 described in Example 1 and the light chain variable region comprises the light chain variable region of L4 described in Example 1.

Note that in Antibodies A-G, lysine (Lys) that is located at the C-terminal of the heavy chain of a common human antibody is deleted.

Example 2 Neutralizing Activity of Anti-Human Notch4 Antibody

The neutralizing activity of anti-human Notch4 antibody (Antibody B) was evaluated with Notch4-GAL4 luciferase reporter assay. This experiment is an experiment system that evaluates signal transduction specific to Notch4 by evaluating the luciferase activity when b.end3 cell line in which a modified gene having a part of Notch4 intracellular domain substituted with GAL4 DNA binding domain as well as a fused gene expression vector between GAL4 UAS and Luciferase 2CP introduced (hereinafter referred as a “reporter cell”) is stimulated with DLL4, which is a Notch ligand.

Recombinant human DLL4 (R&D Systems, 1506-D4-050/CF) was dissolved in PBS to prepare a 10 μg/mL solution (hereinafter DLL4 solution). To a flat-bottomed 96-well white plate (Greiner, 655083), 50 μL/well (500 ng/well) of the DLL4 solution and 50 μL/well of PBS for non-stimulated wells were each dispensed, and this was left overnight at 4° C. to allow DLL4 to be solid phased to the 96-well white plate. The reporter cells were suspended in a D-MEM culture medium comprising 10% Fetal Bovine Serum (FBS) and penicillin/streptomycin to prepare a cell suspension at 1×10^5/mL. Each well with solid phased DLL4 was washed three times with PBS, and 50 μL/well (5,000 cells/well) of the cell suspension was seeded. Anti-human Notch4 antibody dilutions (final concentrations: 0, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10 μg/mL) or Human IgG2 κ (SIGMA, 15404, final concentration: 10 μg/mL) were each added at 50 μL, and this was cultured at 37° C. for 22 hours. The luciferase activity of the reporter cells was evaluated with Steady-Glo Assay System (Promega, E2510) as follows.

One hundred microliters of Steady-Glo solution was added to each well after culturing, stirred, and then left at room temperature for 30 minutes. Luminescence was measured with Multilabel Plate Reader (Envision 2102-0020, Perkin Elmer). Relative luminescence (%) was calculated from the measured luminescence value by the following formula. Relative luminescence (%)=(Luminescence intensity of the specimen well−Average luminescence intensity of non-stimulated wells)/(Average luminescence intensity of control wells−Average luminescence intensity of non-stimulated wells)

The relationship between the concentration of Antibody B and the relative luminescence (%) value is shown in FIG. 1. The graph in FIG. 1 shows the average value of three independent test results, and the error bar shows the standard deviation thereof. IC50 was 0.011 μg/mL (95% CI; 0.0036−0.034).

Next, similar experiments were performed for a plurality of anti-human Notch4 antibodies including Antibody B (Antibody A, Antibody B, Antibody C, Antibody D, Antibody E, Antibody F, and Antibody G), and the neutralizing activity of the antibodies were measured.

Recombinant human DLL4 (R&D Systems, 1506-D4-050/CF) was dissolved in PBS to prepare a 10 μg/mL solution (hereinafter DLL4 solution). To a flat-bottomed 96-well white plate (Greiner, 655083), 50 μL/well (500 ng/well) of the DLL4 solution and 50 μL/well of PBS for non-stimulated wells were each dispensed, and this was left overnight at 4° C. to allow DLL4 to be solid phased to the 96-well white plate. The reporter cells were suspended in a D-MEM culture medium comprising 10% Fetal Bovine Serum (FBS) and penicillin/streptomycin to prepare a cell suspension at 1×10^5/mL. Each well with solid phased DLL4 was washed three times with PBS, and 50 μL/well (5,000 cells/well) of the cell suspension was seeded. Each anti-human Notch4 antibody dilution (final concentrations: 0 and 10 μg/mL) or Human IgG2 κ (SIGMA, 15404, final concentration: 10 μg/mL) was added at 50 μL, and this was cultured at 37° C. for 22 hours. The luciferase activity of the reporter cells was evaluated with Dual luc-Glo Assay System (Promega, E2940) as follows.

One hundred microliters of Dual-Glo Luciferase Substrate solution was added to each well after culturing, stirred, and then left at room temperature for 20 minutes. Luminescence of firefly luciferase was measured with Multilabel Plate Reader (Envision 2102-0020, Perkin Elmer). Next, 100 μL of Dual-Glo Stop & Glo Substrate solution was added to each well, stirred, and then left at room temperature for 20 minutes. Luminescence of Renilla luciferase was measured with Multilabel Plate Reader (Envision 2102-0020, Perkin Elmer). Relative Luminescence of each well (firefly luciferase/Renilla luciferase) was calculated from the ratio of luminescence values. Further, relative luminescence (%) was calculated from each calculated value using the following formula, and Notch4 signal inhibitory activity of each antibody was evaluated. Relative luminescence (%)=(Average relative luminescence of specimen wells−Average relative luminescence of non-stimulated wells)/(Average relative luminescence of control wells−Average relative luminescence of non-stimulated wells)

Notch4 signal inhibitory activity of each antibody (tests performed at 10 μg/ml concentration) is described in Table 28 below.

In the table below, e.g. the description “HK2L3 (Lys-)” means that the heavy chain variable region of the humanized anti-human Notch4 antibody employed in the experiment is the heavy chain variable region of the humanized anti-human Notch4 antibody heavy chain HK2 in Example 1, light chain variable region is the light chain variable region of the humanized anti-human Notch4 antibody light chain L3 in Example 1, and lysine (Lys) located at the C-terminal of the heavy chain of a common human antibody is deleted in this antibody.

TABLE 28 % of Sample H/L chain Control S.D. p value Antibody A HK2L3 (Lys-) 0.1 3.2 5.9E−11 Antibody B HK3L3 (Lys-) 10.8 11.4 8.3E−07 Antibody C HK2L4 (Lys-) −9.9 14.0 8.0E−07 Antibody D HK3L5 (Lys-) 21 8 1.8E−07 Antibody E HK2L6 (Lys-) 5 3 1.7E−11 Antibody F HA2L3 (Lys-) 17 5 3.5E−09 Antibody G HK3L4 (Lys-) 13 9 2.4E−07

Example 3 Kinetic Analysis of Binding of Humanized Anti-Notch4 Antibody to Recombinant Notch4-NRR Domain Protein

Kinetic analysis of the interaction of human, cynomolgus monkey, mouse, and rat Notch4-NRR domains with Antibody B was carried out with BIACORE. Antibody B was purified with protein A affinity chromatography from the culture supernatant of a stable CHO cell line transfected with Antibody B. Human, monkey, mouse, and rat Notch4-NRR domains were prepared as fusion proteins with secretory alkaline phosphatase (SEAP) and 10× histidine tag. The genes for these proteins were transfected into Expi293F cells in Opti-MEM (INVITROGEN) using ExpiFectamine 293. These cells were cultured in Expi293 Expression Medium (INVITROGEN). Briefly, cells were diluted to 7.5×10⁷ cells/25.5 mL, and transfected by ExpiFectamine 293 reagent on Day 0. About 16 hours after transfection, 150 uL of ExpiFectamine 293 Transfection Enhancer 1 and 1.5 mL of ExpiFectamine 293 Transfection Enhancer 2 were added to each flask. The supernatant was collected on Day 4. These antigens were purified with Ni-NTA Superflow column (QIAGEN). The interaction was analyzed as below. The purified Antibody B was captured by anti-human IgG Fc antibody fixed on a CM5 sensor chip (GE healthcare). The purified Notch4-NRR fusion proteins were injected onto the sensor chip at 8 different concentrations, and the interaction and dissociation thereof were observed as per manufacturer's instructions.

Table 29 Calculated Kinetic Parameters of Antibody B Protein Ka (1/Ms) Kd (1/s) KD (M) Human Notch4-NRR 3.17E+05 1.18E−03 3.74E−09 Cynomolgus monkey 2.95E+05 1.06E−03 3.62E−09 Notch4-NRR Mouse Notch4-NRR 6.01E+05 7.27E−03 1.12E−08 Rat Notch4-NRR ND ND ND

Kinetic analysis of further anti-Notch4 inhibitory humanized antibodies was carried out as follows. Human Notch4-NRR domain Fc fusion proteins were expressed with a CHO cell line. The antigen in the culture supernatant was captured by the anti-human Notch4 antibody fixed on a CM5 sensor chip that recognizes different epitopes of human Notch4. The humanized anti-human Notch4 antibody was then injected onto the sensor chip at various concentrations. The interaction and disassociation constants thereof were calculated according to manufacturer's instructions. The results are shown in Table 30.

In the table below, e.g. the description “HK2L3 (Lys-)” means that the heavy chain variable region of the humanized anti-human Notch4 antibody employed in the experiment is the heavy chain variable region of the humanized anti-human Notch4 antibody heavy chain HK2 in Example 1, light chain variable region is the light chain variable region of the humanized anti-human Notch4 antibody light chain L3 in Example 1, and lysine (Lys) located at the C-terminal of the heavy chain of a common human antibody is deleted in this antibody. Those without the description “(Lys-)” mean that Lys located at the C-terminal of the heavy chain of the human antibody is not deleted.

TABLE 30 Calculated Kinetic Parameters of Humanized Anti-Notch4 Inhibitory Antibodies Against Human Notch4-NRR Fusion Protein Rmax1 Sample H/L Chain ka (1/Ms) kd (1/s) KD (M) (RU) Antibody A HK2L3(Lys-) 1.62E+06 8.89E−04 5.55E−10 33.4 Antibody B HK3L3(Lys-) 1.66E+06 7.10E−04 4.41E−10 26.2 Antibody C HK2L4(Lys-) 1.91E+06 1.06E−03 5.56E−10 35.9 Antibody D HK3L5(Lys-) 1.82E+06 8.63E−04 4.75E−10 37 Antibody E HK2L6(Lys-) 2.23E+06 1.15E−03 5.19E−10 28.9 Antibody F HA2L3(Lys-) 1.55E+06 1.36E−03 9.82E−10 20.2 Antibody G HK3L4(Lys-) 1.78E+06 1.11E−03 6.33E−10 29.2

Experimental results of experiments similar to the above Table 30 performed with further more antibodies are shown in Table 31.

TABLE 31 ka kd kD No. HL construct (averaged) (averaged) (averaged) N 6-3-A6 Original 2.35E+06 7.42E−04 3.21E−10 9 37 HK3L3(Lys-) 2.48E+06 8.22E−04 3.31E−10 2 34 HK2L3(Lys-) 5.05E+06 1.24E−03 3.95E−10 2 31 HA2L3(Lys-) 3.50E+06 8.94E−04 4.34E−10 2 39 HK3L5(Lys-) 2.10E+06 1.09E−03 5.29E−10 5 15 HK2L5 2.07E+06 1.12E−03 5.49E−10 6 20 HK3L5 1.72E+06 1.02E−03 6.01E−10 6 30 HA2L5 2.49E+06 1.36E−03 6.12E−10 6 36 HK2L5(Lys-) 1.93E+06 1.13E−03 6.17E−10 5 41 HK2L6(Lys-) 1.87E+06 1.15E−03 6.20E−10 3 14 HK2L4 1.89E+06 1.17E−03 6.34E−10 6 13 HK2L3 1.62E+06 1.09E−03 6.82E−10 6 35 HK2L4(Lys-) 1.81E+06 1.27E−03 7.15E−10 5 28 HA2L3 1.95E+06 1.37E−03 7.27E−10 6 29 HA2L4 2.33E+06 1.69E−03 7.38E−10 6 19 HK3L4 1.61E+06 1.20E−03 7.55E−10 6 38 HK3L4(Lys-) 1.86E+06 1.24E−03 7.60E−10 5 42 HK3L6(Lys-) 1.51E+06 1.14E−03 7.66E−10 3 33 HA2L5(Lys-) 2.01E+06 1.50E−03 7.76E−10 3 32 HA2L4(Lys-) 2.43E+06 1.62E−03 8.30E−10 5 40 HA2L6(Lys-) 1.83E+06 1.51E−03 8.38E−10 3 18 HK3L3 1.22E+06 1.04E−03 8.97E−10 6 17 HK3L2 1.13E+06 1.45E−03 1.29E−09 4 12 HK2L2 1.23E+06 1.57E−03 1.38E−09 4 27 HA2L2 1.63E+06 2.22E−03 1.41E−09 4 16 HK3L1 7.83E+05 1.33E−03 1.76E−09 4 26 HA2L1 9.49E+05 1.66E−03 1.76E−09 4 10 HK1L5 2.72E+06 4.89E−03 1.78E−09 3 11 HK2L1 8.75E+05 1.60E−03 1.84E−09 4 9 HK1L4 2.78E+06 1.18E−02 4.34E−09 2 8 HK1L3 3.71E+06 1.96E−02 4.87E−09 2 25 HA1L5 6.36E+06 4.52E−02 7.20E−09 2 24 HA1L4 1.18E+07 1.13E−01 9.54E−09 1 23 HA1L3 3.57E+07 5.24E−01 1.47E−08 1 22 HA1L2 6.49E+05 1.22E−02 1.86E−08 2 21 HA1L1 6.15E+04 2.79E−02 4.53E−07 1

Example 4 In Vivo Pharmacological Test Employing Anti-Human Notch4 Antibody (Antitumor Effect and Blood Perfusion Suppressive Effect of Antibody B in Calu6 Xenograft Model)

Human non-small cell lung cancer cell line Calu6 (ATCC number HTB-56) cultured in an EMEM culture medium comprising 10% FBS, MEM non-essential amino acids, sodium pyruvate, and penicillin/streptomycin was prepared to a concentration of 1.6×10⁸ cells/mL with EMEM culture medium, and mixed with Matrigel™ (CORNING Cat#354234) at 1:1 to prepare a cell suspension of 8.0×10⁷ cells/mL. A dose of 0.1 mL was subcutaneously transplanted to the right posterior dorsal region of 6 weeks-old nude mice (CAnN.Cg-Foxn1nu/CrlCrlj, female, CHARLES RIVER LABORATORIES JAPAN, INC.) Twenty-eight days after transplantation, minor and major axes of the tumor were measured with an electronic digital caliper (DIGIMATIC™ CALIPER, Mitsutoyo Corporation), and the tumor volume TV was calculated with the following calculation formula. TV (mm³)=Major axis (mm)×Minor axis (mm)×Minor axis (mm)/2

Randomization was carried out based on the tumor volume on the first day of administration so that the average value of the tumor volumes will be approximately equal among the groups. Antibody B was diluted immediately before administration so that vehicle solution (25 mM Histidine, 250 mM Sucrose, and 0.05% Tween80 (pH5.3)) and saline is 1:9 to obtain 0.1, 0.3, and 1.0 mg/mL evaluation specimens (1, 3, and 10 mg/kg administration group, respectively). Evaluation specimens were administered by tail vein injection at a dosage of 0.2 mL/20 g of mouse body weight twice a week (Day 1 and Day 4 when counting the day of grouping as Day 1). For the control group, 11.6 mg/mL of Control IgG (ChromPure Human IgG, whole molecule, Jackson ImmunoResearch Laboratories, Cat#009-000-003) was diluted with PBS to 1.0 mg/mL, and administered by tail vein injection at 0.2 mL/20 g of mouse body weight (administration volume 10 mg/kg). The experiment was performed with 8 mice per group. Relative tumor volume RTV was calculated for each of the Control IgG group and Antibody B administration group with the following formula and shown in FIG. 2A. RTV=Tumor volume on the day of measurement/Tumor volume at the start of administration

Antibody B showed significant antitumor effect at all dosages in Calu6 xenograft model.

Tumor blood perfusion was evaluated by determining the fluorescence by nucleus staining of cells around blood vessels due to a fluorescent dye (Hoechst) injected in the tail vein (Funahashi et al. (2014), Cancer Sci., 105(10), 1334-42.). After measuring the tumor diameter on the final test day, 10 mg/mL of Hoechst 33342 (Life technologies Cat# H3570) was diluted to 2× with PBS, and 5.0 mg/mL of the diluted Hoechst 33342 was injected in the tail vein at 0.1 mL/head. Mice were euthanized by cervical dislocation 5 minutes after injecting Hoechst, and the collected tumors were embedded in OCT Compound (Sakura Finetek Japan Co., Ltd. Cat#4583) to produce frozen blocks.

Tumor blood vessels of frozen sections were subjected to immunofluorescent staining with anti-CD31 antibody (FITC conjugated, BD Pharmingen Cat#553372), and Hoechst fluorescence of tumor blood vessel hotspots (5 per tumor) were photographed with BIOREVO fluorescence microscope (KEYENCE, BZ-9000). Hoechst-positive area was determined with an image analysis software (Lumina Vision ver 2.2.2, MITANI CORPORATION), and the average of each tumor section was calculated and shown in FIG. 2B.

Antibody B showed significant blood perfusion suppressive effect at all dosages in Calu6 xenograft model.

Example 5 Combination Use of Antibody B and Cisplatin in Calu6 Xenograft Model

Human non-small cell lung cancer cell line Calu6 (ATCC number HTB-56) cultured in an EMEM culture medium comprising 10% FBS and penicillin/streptomycin was prepared to a concentration of 1.2×10⁸ cells/mL with EMEM culture medium, and mixed with Matrigel™ (CORNING Cat#354234) at 1:1 to prepare a cell suspension of 6.0×10⁷ cells/mL. A dose of 0.1 mL was subcutaneously transplanted to the right posterior dorsal region of 7 weeks-old nude mice (CAnN.Cg-Foxn1nu/CrlCrlj, female, CHARLES RIVER LABORATORIES JAPAN, INC.) Twenty-seven days after transplantation, minor and major axes of the tumor were measured with an electronic digital caliper (DIGIMATIC™ CALIPER, Mitsutoyo Corporation), and the tumor volume TV was calculated with the following calculation formula. Tumor volume TV (mm³)=Major axis (mm)×Minor axis (mm)×Minor axis (mm)/2

Randomization was carried out based on the tumor volume on the first day of administration so that the average value of tumor volumes will be approximately equal among the groups. For Antibody B, a 10.34 mg/mL solution (vehicle: 25 mM Phosphate, 200 mM Trehalose, and 0.05% Tween80 (pH 5.5)) was diluted immediately before administration with PBS to prepare a solution at 2.5 mg/mL, and this was administered by intravenous injection at a dosage of 0.2 mL (500 μg)/head, twice a week for 5 weeks. Cisplatin was administered once on the first day of administration by tail vein administration at a dose of 10 mg/kg. The experiment was performed with 4 mice per group. The relative tumor volume RTV was calculated for each of the control (non-treated) group, Antibody B administration group, cisplatin administration group, and Antibody B+cisplatin combination group with the following formula and shown in FIG. 3. RTV=Tumor volume on the day of measurement/Tumor volume at the start of administration

The combination use of Antibody B and cisplatin showed significantly superior antitumor effect compared to cisplatin alone in Calu6 xenograft model.

Example 6 Combination Use of Antibody B and Lenvatinib Mesylate in FTC238 Xenograft Model

Human thyroid cancer cell line FTC238 (purchased from Sumitomo Dainippon Pharma Co., Ltd.) cultured in an DMEM/HAM's F12 (1:1) culture medium comprising 10% FBS and penicillin/streptomycin was prepared to a concentration of 1.2×10⁸ cells/mL with DMEM/HAM's F12 (1:1) culture medium, and mixed with Matrigel™ (CORNING Cat#354234) at 1:1 to prepare a cell suspension of 6.0×10⁷ cells/mL. A dose of 0.1 mL was subcutaneously transplanted to the right flank of 7 weeks-old nude mice (CAnN.Cg-Foxn1nu/CrlCrlj, female, CHARLES RIVER LABORATORIES JAPAN, INC.) Eight days after transplantation, minor and major axes of the tumor were measured with an electronic digital caliper (DIGIMATIC™ CALIPER, Mitsutoyo Corporation), and the tumor volume TV was calculated with the following calculation formula. Tumor volume TV (mm³)=Major axis (mm)×Minor axis (mm)×Minor axis (mm)/2

Randomization was carried out based on the tumor volume on the first day of administration so that the average value of tumor volumes will be approximately equal among the groups. For Antibody B, 10.8 mg/mL Antibody B (vehicle: 25 mM Histidine and 250 mM Sucrose (pH 5.3)) was diluted immediately before administration with the vehicle solution to prepare 2.5 mg/mL Antibody B, and this was administered by tail vein injection at a dosage of 0.2 mL (500 μg)/head, twice a week for 2 weeks. Lenvatinib mesylate was orally administered at a dose of 10 mg/kg, once a day for 12 days. The experiment was performed with 5 mice per group. The relative tumor volume RTV was calculated for each of the control (non-treated) group, Antibody B administration group, lenvatinib mesylate administration group, and Antibody B+lenvatinib mesylate combination group with the following formula and shown in FIG. 4. RTV=Tumor volume on the day of measurement/Tumor volume at the start of administration

The combination use of Antibody B and lenvatinib mesylate showed significantly superior antitumor effect compared to Antibody B administered alone or lenvatinib mesylate alone in FTC238 xenograft model.

Example 7 Combination Use of Mouse Antibody 6-3-A6 and Paclitaxel in DU145 Xenograft Model

Human prostate cancer cell line DU145 (ATCC number HTB-81) cultured in a RPMI1640 culture medium comprising 10% FBS, sodium pyruvate, 2-mercaptoethanol, and penicillin/streptomycin was prepared to a concentration of 6.0×10⁷ cells/mL with RPMI1640 culture medium. A dose of 0.1 mL was subcutaneously transplanted to the right flank of 6 weeks-old nude mice (CAnN.Cg-Foxn1nu/CrlCrlj, female, CHARLES RIVER LABORATORIES JAPAN, INC.) Twenty-four days after transplantation, minor and major axes of the tumor were measured with an electronic digital caliper (DIGIMATIC™ CALIPER, Mitsutoyo Corporation), and the tumor volume TV was calculated with the following calculation formula. Tumor volume TV (mm³)=Major axis (mm)×Minor axis (mm)×Minor axis (mm)/2

Randomization was carried out based on the tumor volume on the first day of administration so that the average value of tumor volumes will be approximately equal among the groups. For 6-3-A6, 5.11 mg/mL 6-3-A6 (vehicle: PBS) was diluted immediately before administration with PBS to prepare 2.5 mg/mL 6-3-A6, and this was administered by tail vein injection at a dosage of 0.2 mL (500 μg)/head, twice a week for 4 weeks. Paclitaxel was administered by tail vein administration at a dose or 20 mg/kg, once a day for 5 days. The experiment was performed with 4 mice per group. The tumor volume TV of each of the control (non-treated) group, 6-3-A6 administration group, paclitaxel administration group, and 6-3-A6+paclitaxel combination group are shown in FIG. 5.

The combination use of 6-3-A6 and paclitaxel showed significantly superior antitumor effect compared to 6-3-A6 alone or paclitaxel alone in DU145 xenograft model.

Example 8 Combination of Antibody B with Lenvatinib Mesilate in Hepatocellular Carcinoma Patient Derived Xenograft Model

To examine the antitumor effect of Antibody B on hepatocellular carcinoma (HCC), LI0050, a HuPrime® patient derived xenograft model (Crown Bioscience Inc.), was used. The LI0050 is a model mouse inoculated with primary tumor tissues from a female HCC patient, which has been reported sorafenib resistant (International Patent Pamphlet WO2015/031604).

Tumor fragments from LI0050 stock mice were harvested and a fragment of 2-4 mm in diameter was inoculated subcutaneously into the right flank of BALB/c nude mice for tumor development. Tumor size was measured in two dimensions using a caliper, and the tumor volume TV was calculated with the following calculation formula. Tumor volume TV (mm³)=Major axis (mm)×Minor axis (mm)×Minor axis (mm)/2

The treatment was started when the average tumor size reached about 192 mm³. Randomization was carried out based on the tumor volume on the first day of administration so that the average value of tumor volumes will be approximately equal among the groups. Each group consisted of 10 mice. The day of randomization was denoted as Day 0. From Day 0 through Day 13, mice of each group were treated once daily by each of (i) control (3 mM HCl), (ii) lenvatinib mesilate (10 mg/kg), (iii) sorafenib tosylate (30 mg/kg), or (iv) lenvatinib mesilate (10 mg/kg) plus Antibody B (0.5 mg/mouse).

For comparison between two groups, an independent sample t-test has been used.

The combination of Antibody B and lenvatinib mesilate showed significantly superior antitumor effect compared with control group.

Example 9 Comparison of Signal Inhibitory Activity Between Anti-Human Notch4 Polyclonal Antibody and Antibody B

Next, signal inhibitory activities of polyclonal anti-human Notch4 antibody (Santa Cruz, SC8643, hereinafter N-17) and Antibody B were compared with Notch4-GAL4 luciferase reporter assay system.

Slide-A-Lyzer (Thermo scientific, 66333) was employed to perform dialysis at 4° C. for 8 hours in PBS in order to remove sodium azide contained in N-17. After concentrating the dialyzed N-17 solution with Amicon Ultra (Millipore, UFC503096), and the concentration was measured with a microspectrophotometer (Nano Drop, Thermo).

Recombinant human DLL4 (R&D Systems, 1506-D4-050/CF) was dissolved in PBS to prepare a 10 μg/mL solution (hereinafter DLL4 solution). To a flat-bottomed 96 well white plate (Greiner, 655083), 50 μL/well (500 ng/well) of the DLL4 solution and 50 μL/well of PBS for non-stimulated wells were each dispensed, and this was left overnight at 4° C. to allow DLL4 to be solid phased to the 96-well white plate. The reporter cells were suspended in a D-MEM culture medium comprising 10% Fetal Bovine Serum (FBS) and penicillin/streptomycin to prepare a cell suspension at 1×10^5/mL. Each well with solid phased DLL4 was washed three times with PBS, and 50 μL/well (5,000 cells/well) of the cell suspension was seeded. After dialysis/concentration, N-17 dilutions or Antibody B dilutions diluted with the culture medium (final concentrations: 0, 0.01, 0.1, 1, and 10 μg/mL) were each added at 50 μL, and this was cultured at 37° C. for 22 hours. The luciferase activity of the reporter cells were evaluated with Steady-Glo Assay System (Promega, E2510) as follows.

One hundred microliters of Steady-Glo solution was added to each well after culturing, stirred, and then left at room temperature for 30 minutes. Luminescence was measured with Multilabel Plate Reader (Envision 2102-0020, Perkin Elmer). Relative luminescence was calculated from the measured luminescence value by the following formula. Relative luminescence (%)=(Luminescence intensity of the specimen well−Average luminescence intensity of non-stimulated wells)/(Average luminescence intensity of control wells−Average luminescence intensity of non-stimulated wells)

The relationship between the concentration of Antibody B and the relative luminescence (%) value is shown in FIG. 7. The graph in FIG. 7 shows the average value of three independent test results, and the error bar shows the standard deviation thereof. Signal inhibitory activity was confirmed for Antibody B, but for N-17, Notch4 signal inhibitory activity of N-17 was not seen in the investigated concentration range.

Example 10 Kinetics Analysis of Binding Between Antibody B and Recombinant Soluble Human Notch NRR Domain

Kinetics analysis using BIAcore T100 was performed for the interaction between the NRR domain of human Notch isotypes (Notch1, Notch2, Notch3, and Notch4) and Antibody B. Antibody B was purified from the culture supernatant of a stable expression CHO cell line of Antibody B with sequential use of protein A affinity chromatography, Capto Q anion exchange chromatography, and UNOsphere S cation exchange chromatography. Fusion proteins of Human Notch1 NRR domain (Genbank Accession No. 017617; sequence positions 1307-1733), human Notch2 NRR domain (Genbank Accession No. 024408; sequence positions 1239-1650), human Notch3 NRR domain (Genbank Accession No. 000435; sequence positions 1246-1641), and human Notch4 NRR domain (Genbank Accession No. NP_004548.3; sequence positions 1046-1445) with secretory alkaline phosphatase (SEAP), a hemagglutinin (HA) tag, and a histidine tag (×10) were created, and these were purified with HisTrap™ Fast Flow column (GE Healthcare). The interaction between Antibody B and the NRR domain of each human Notch isotypes was measured with the following method. The purified Antibody B was captured by anti-human IgG Fc antibody fixed on a CM5 sensor chip (GE Healthcare). The purified NRR domain of each human Notch isotypes was injected onto the sensor chip at 6 different concentrations, and the interaction and dissociation with the antibody were observed as per operation manual.

The overlaid interaction sensorgram and the calculated kinetics parameters are each shown in FIGS. 8A-8D and Table 32.

TABLE 32 Calculated Kinetic Parameters of Interaction Between Antibody B and Human Notch4-NRR Fusion Proteins Sample Ka (1/Ms) Kd (us) KD (M) Human ND ND ND Notch1-NRR-SEAP-HA-His Human ND ND ND Notch2-NRR-SEAP-HA-His Human ND ND ND Notch3-NRR-SEAP-HA-His Human 2.72E+04 8.31E−04 3.05E−08 Notch4-NRR-SEAP-HA-His 

The invention claimed is:
 1. An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or Notch4 binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45, (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45, (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47, (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:49, (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:51, (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45, or (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47.
 2. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45.
 3. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47.
 4. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:49.
 5. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:51.
 6. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45.
 7. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47.
 8. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 1, comprising a heavy chain constant region comprising a sequence derived from a human antibody and a light chain constant region comprising a sequence derived from a human antibody.
 9. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 8, wherein the heavy chain constant region comprises the constant region of a human IgG.
 10. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 9, wherein the human IgG is human IgG2.
 11. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 8, wherein the heavy chain constant region comprises the constant region of human IgG2 with the substitution V234A, G237A, or V234A and G237A.
 12. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 8, wherein the lysine residue at the carboxy terminal of the heavy chain constant region is artificially removed.
 13. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 8, wherein the light chain constant region comprises the constant region of human Igκ.
 14. A pharmaceutical composition comprising the anti-Notch4 antibody or Notch4 binding fragment thereof according to claim
 8. 15. The pharmaceutical composition according to claim 14 which further comprises a pharmaceutically acceptable carrier.
 16. A method of treating non-small cell lung cancer, thyroid cancer, prostate cancer or hepatocellular carcinoma in a human subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 14 to the human subject.
 17. An anti-Notch4 antibody or a Notch4 binding fragment thereof, wherein said antibody or Notch4 binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:35 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:45.
 18. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 17, comprising a heavy chain constant region comprising a sequence derived from a human antibody and a light chain constant region comprising a sequence derived from a human antibody.
 19. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 18, wherein the heavy chain constant region comprises the constant region of a human IgG.
 20. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 19, wherein the human IgG is human IgG2.
 21. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 18, wherein the heavy chain constant region comprises the constant region of human IgG2 with the substitution V234A, G237A, or V234A and G237A.
 22. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 18, wherein the lysine residue at the carboxy terminal of the heavy chain constant region is artificially removed.
 23. The anti-Notch4 antibody or Notch4 binding fragment thereof according to claim 18, wherein the light chain constant region comprises the constant region of human Igκ.
 24. A pharmaceutical composition comprising the anti-Notch4 antibody or Notch4 binding fragment thereof according to claim
 18. 25. The pharmaceutical composition according to claim 24 which further comprises a pharmaceutically acceptable carrier.
 26. A method of treating non-small cell lung cancer carcinoma in a human subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 24 to the human subject.
 27. A method of treating thyroid cancer in a human subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 24 to the human subject.
 28. A method of treating prostate cancer in a human subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 24 to the human subject.
 29. A method of treating hepatocellular carcinoma in a human subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 24 to the human subject. 